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Sample records for reveals unexpected complexity

  1. High-resolution molecular genomic autopsy reveals complex sudden unexpected death in epilepsy risk profile.

    PubMed

    Klassen, Tara L; Bomben, Valerie C; Patel, Ankita; Drabek, Janice; Chen, Tim T; Gu, Wenli; Zhang, Feng; Chapman, Kevin; Lupski, James R; Noebels, Jeffrey L; Goldman, A M

    2014-02-01

    Advanced variant detection in genes underlying risk of sudden unexpected death in epilepsy (SUDEP) can uncover extensive epistatic complexity and improve diagnostic accuracy of epilepsy-related mortality. However, the sensitivity and clinical utility of diagnostic panels based solely on established cardiac arrhythmia genes in the molecular autopsy of SUDEP is unknown. We applied the established clinical diagnostic panels, followed by sequencing and a high density copy number variant (CNV) detection array of an additional 253 related ion channel subunit genes to analyze the overall genomic variation in a SUDEP of the 3-year-old proband with severe myoclonic epilepsy of infancy (SMEI). We uncovered complex combinations of single nucleotide polymorphisms and CNVs in genes expressed in both neurocardiac and respiratory control pathways, including SCN1A, KCNA1, RYR3, and HTR2C. Our findings demonstrate the importance of comprehensive high-resolution variant analysis in the assessment of personally relevant SUDEP risk. In this case, the combination of de novo single nucleotide polymorphisms (SNPs) and CNVs in the SCN1A and KCNA1 genes, respectively, is suspected to be the principal risk factor for both epilepsy and premature death. However, consideration of the overall biologically relevant variant complexity with its extensive functional epistatic interactions reveals potential personal risk more accurately. PMID:24372310

  2. Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans

    PubMed Central

    Fraune, Johanna; Alsheimer, Manfred; Volff, Jean-Nicolas; Busch, Karoline; Fraune, Sebastian; Bosch, Thomas C. G.; Benavente, Ricardo

    2012-01-01

    The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed no sequence homology. This discrepancy challenged the hypothesis that the SC arose only once in evolution. To pursue this matter we focused on the evolution of SYCP1 and SYCP3, the two major structural SC proteins of mammals. Remarkably, our comparative bioinformatic and expression studies revealed that SYCP1 and SYCP3 are also components of the SC in the basal metazoan Hydra. In contrast to previous assumptions, we therefore conclude that SYCP1 and SYCP3 form monophyletic groups of orthologous proteins across metazoans. PMID:23012415

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

    PubMed

    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. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26537438

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

    PubMed

    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. PMID:26253310

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

    PubMed

    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. PMID:20707997

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

    PubMed Central

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

    2010-01-01

    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 reorganization 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 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. PMID:20707997

  7. Structural Analysis of the Maize Rp1 Complex Reveals Numerous Sites and Unexpected Mechanisms of Local Rearrangement

    PubMed Central

    Ramakrishna, Wusirika; Emberton, John; Ogden, Matthew; SanMiguel, Phillip; Bennetzen, Jeffrey L.

    2002-01-01

    Rp1 is a complex disease resistance locus in maize that is exceptional in both allelic variability and meiotic instability. Genomic sequence analysis of three maize BACs from the Rp1 region of the B73 inbred line revealed 4 Rp1 homologs and 18 other gene-homologous sequences, of which at least 16 are truncated. Thirteen of the truncated genes are found in three clusters, suggesting that they arose from multiple illegitimate break repairs at the same sites or from complex repairs of each of these sites with multiple unlinked DNA templates. A 43-kb region that contains an Rp1 homolog, six truncated genes, and three Opie retrotransposons was found to be duplicated in this region. This duplication is relatively recent, occurring after the insertion of the three Opie elements. The breakpoints of the duplication are outside of any genes or identified repeat sequence, suggesting a duplication mechanism that did not involve unequal recombination. A physical map and partial sequencing of the Rp1 complex indicate the presence of 15 Rp1 homologs in regions of ∼250 and 300 kb in the B73 inbred line. Comparison of fully sequenced Rp1-homologous sequences in the region demonstrates a history of unequal recombination and diversifying selection within the Leu-rich repeat 2 region, resulting in chimeric gene structures. PMID:12468738

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

    USGS Publications Warehouse

    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

  9. Dissecting tRNA-derived fragment complexities using personalized transcriptomes reveals novel fragment classes and unexpected dependencies

    PubMed Central

    Telonis, Aristeidis G.; Loher, Phillipe; Honda, Shozo; Jing, Yi; Palazzo, Juan; Kirino, Yohei; Rigoutsos, Isidore

    2015-01-01

    We analyzed transcriptomic data from 452 healthy men and women representing five different human populations and two races, and, 311 breast cancer samples from The Cancer Genome Atlas. Our studies revealed numerous constitutive, distinct fragments with overlapping sequences and quantized lengths that persist across dozens of individuals and arise from the genomic loci of all nuclear and mitochondrial human transfer RNAs (tRNAs). Surprisingly, we discovered that the tRNA fragments' length, starting and ending points, and relative abundance depend on gender, population, race and also on amino acid identity, anticodon, genomic locus, tissue, disease, and disease subtype. Moreover, the length distribution of mitochondrially-encoded tRNAs differs from that of nuclearly-encoded tRNAs, and the specifics of these distributions depend on tissue. Notably, tRNA fragments from the same anticodon do not have correlated abundances. We also report on a novel category of tRNA fragments that significantly contribute to the differences we observe across tissues, genders, populations, and races: these fragments, referred to as i-tRFs, are abundant in human tissues, wholly internal to the respective mature tRNA, and can straddle the anticodon. HITS-CLIP data analysis revealed that tRNA fragments are loaded on Argonaute in a cell-dependent manner, suggesting cell-dependent functional roles through the RNA interference pathway. We validated experimentally two i-tRF molecules: the first was found in 21 of 22 tested breast tumor and adjacent normal samples and was differentially abundant between health and disease whereas the second was found in all eight tested breast cancer cell lines. PMID:26325506

  10. Sequence tagging reveals unexpected modifications in toxicoproteomics

    PubMed Central

    Dasari, Surendra; Chambers, Matthew C.; Codreanu, Simona G.; Liebler, Daniel C.; Collins, Ben C.; Pennington, Stephen R.; Gallagher, William M.; Tabb, David L.

    2010-01-01

    Toxicoproteomic samples are rich in posttranslational modifications (PTMs) of proteins. Identifying these modifications via standard database searching can incur significant performance penalties. Here we describe the latest developments in TagRecon, an algorithm that leverages inferred sequence tags to identify modified peptides in toxicoproteomic data sets. TagRecon identifies known modifications more effectively than the MyriMatch database search engine. TagRecon outperformed state of the art software in recognizing unanticipated modifications from LTQ, Orbitrap, and QTOF data sets. We developed user-friendly software for detecting persistent mass shifts from samples. We follow a three-step strategy for detecting unanticipated PTMs in samples. First, we identify the proteins present in the sample with a standard database search. Next, identified proteins are interrogated for unexpected PTMs with a sequence tag-based search. Finally, additional evidence is gathered for the detected mass shifts with a refinement search. Application of this technology on toxicoproteomic data sets revealed unintended cross-reactions between proteins and sample processing reagents. Twenty five proteins in rat liver showed signs of oxidative stress when exposed to potentially toxic drugs. These results demonstrate the value of mining toxicoproteomic data sets for modifications. PMID:21214251

  11. Managing the Unexpected: Complexity as Distributed Sensemaking

    NASA Astrophysics Data System (ADS)

    Weick, Karl E.

    In 1998 the Centers for Disease Control (CDC) published a statement of their strategy entitled "Preventing Emerging Infectious Diseases: A Strategy for the 21st Century." They described their central challenge this way: "because we do not know what new diseases will arise, we must always be prepared for the unexpected" (p. vii). Soon after they published that statement CDC was confronted with an unexpected emerging disease, the West Nile Virus, which they misdiagnosed initially.

  12. Social performance reveals unexpected vocal competency in young songbirds

    PubMed Central

    Kojima, Satoshi; Doupe, Allison J.

    2011-01-01

    Vocal ontogeny in songbirds provides a good model for understanding how complex motor behavior, including speech, is learned. For birdsong, as for other motor learning, it has generally been assumed that a subject's motor output at any point during learning represents what the subject has learned to produce by that time. Here, we show, however, that juvenile zebra finches partway through song learning, singing immature song, are capable of producing song with much more mature properties, depending on the behavioral context. In these birds, we were able to elicit courtship (female-directed) song, which young birds normally sing infrequently, and to compare it with the alone or “undirected” song (Undir) predominantly produced during learning as well as with the same bird's subsequent adult song. We found that the juvenile courtship song was much less variable than the immature Undir and as stereotyped as the adult song produced after a further month of practice. More strikingly, the juvenile courtship song was also acoustically much more similar than Undir to the adult song. This finding demonstrates that the Undir that juvenile birds usually produce underestimates the extent of learning and that song structure is learned faster than previously thought. Moreover, the rapid improvement in song quality in response to external social cues supports the idea that courtship singing is a state of motor “performance,” in which the bird selects the best variants of the song learned during singing alone, and suggests that such performance states can reveal unappreciated progression of learning. PMID:21220335

  13. Metagenomic Analysis Reveals Unexpected Subgenomic Diversity of Magnetotactic Bacteria within the Phylum Nitrospirae ▿ †

    PubMed Central

    Lin, Wei; Jogler, Christian; Schüler, Dirk; Pan, Yongxin

    2011-01-01

    A targeted metagenomic approach was applied to investigate magnetotactic bacteria (MTB) within the phylum Nitrospirae in Lake Miyun near Beijing, China. Five fosmids containing rRNA operons were identified. Comparative sequence analysis of a total of 172 kb provided new insights into their genome organization and revealed unexpected subgenomic diversity of uncultivated MTB in the phylum Nitrospirae. In addition, affiliation of two novel MTB with the phylum Nitrospirae was verified by fluorescence in situ hybridization. One of them was morphologically similar to “Candidatus Magnetobacterium bavaricum,” but the other differed substantially in cell shape and magnetosome organization from all previously described “Ca. Magnetobacterium bavaricum”-like bacteria. PMID:21057016

  14. The Crystal Structures of EAP Domains from Staphylococcus aureus Reveal an Unexpected Homology to Bacterial Superantigens

    SciTech Connect

    Geisbrecht, B V; Hamaoka, B Y; Perman, B; Zemla, A; Leahy, D J

    2005-10-14

    The Eap (extracellular adherence protein) of Staphylococcus aureus functions as a secreted virulence factor by mediating interactions between the bacterial cell surface and several extracellular host proteins. Eap proteins from different Staphylococcal strains consist of four to six tandem repeats of a structurally uncharacterized domain (EAP domain). We have determined the three-dimensional structures of three different EAP domains to 1.8, 2.2, and 1.35 {angstrom} resolution, respectively. These structures reveal a core fold that is comprised of an {alpha}-helix lying diagonally across a five-stranded, mixed {beta}-sheet. Comparison of EAP domains with known structures reveals an unexpected homology with the C-terminal domain of bacterial superantigens. Examination of the structure of the superantigen SEC2 bound to the {beta}-chain of a T-cell receptor suggests a possible ligand-binding site within the EAP domain (Fields, B. A., Malchiodi, E. L., Li, H., Ysern, X., Stauffacher, C. V., Schlievert, P. M., Karjalainen, K., and Mariuzza, R. (1996) Nature 384, 188-192). These results provide the first structural characterization of EAP domains, relate EAP domains to a large class of bacterial toxins, and will guide the design of future experiments to analyze EAP domain structure/function relationships.

  15. Kinase inhibitor profiling reveals unexpected opportunities to inhibit disease-associated mutant kinases

    PubMed Central

    Duong-Ly, Krisna C.; Devarajan, Karthik; Liang, Shuguang; Horiuchi, Kurumi Y.; Wang, Yuren; Ma, Haiching; Peterson, Jeffrey R.

    2016-01-01

    Summary Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant, mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases including ALK, LRRK2, RET, and EGFR as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development. PMID:26776524

  16. Novel 3D Microscopic Analysis of Human Placental Villous Trees Reveals Unexpected Significance of Branching Angles

    PubMed Central

    Haeussner, Eva; Buehlmeyer, Antonia; Schmitz, Christoph; von Koch, Franz Edler; Frank, Hans-Georg

    2014-01-01

    The villous trees of human placentas delineate the fetomaternal border and are complex three-dimensional (3D) structures. Thus far, they have primarily been analyzed as thin, two-dimensional (2D) histological sections. However, 2D sections cannot provide access to key aspects such as branching nodes and branch order. Using samples taken from 50 normal human placentas at birth, in the present study we show that analysis procedures for 3D reconstruction of neuronal dendritic trees can also be used for analyzing trees of human placentas. Nodes and their branches (e.g., branching hierarchy, branching angles, diameters, and lengths of branches) can be efficiently measured in whole-mount preparations of isolated villous trees using high-end light microscopy. Such data differ qualitatively from the data obtainable from histological sections and go substantially beyond the morphological horizon of such histological data. Unexpectedly, branching angles of terminal branches of villous trees varied inversely with the fetoplacental weight ratio, a widely used clinical parameter. Since branching angles have never before been determined in the human placenta, this result requires further detailed studies in order to fully understand its impact. PMID:25155961

  17. Metatranscriptome Analysis of Aquifer Samples Reveals Unexpected Metabolic Lifestyles Relevant to Active Biogeochemical Cycling

    NASA Astrophysics Data System (ADS)

    Beller, H. R.; Jewell, T. N. M.; Karaoz, U.; Banfield, J. F.; Brodie, E.; Williams, K. H.

    2015-12-01

    Modern molecular ecology techniques are revealing the metabolic potential of uncultivated microorganisms, but there is still much to be learned about the actual biogeochemical roles of microbes that have cultivated relatives. Here, we present metatranscriptomic and metagenomic data from a field study that provides evidence of coupled redox processes that have not been documented in cultivated relatives and, indeed, represent strains with metabolic traits that are novel with respect to closely related isolates. The data come from omics analysis of groundwater samples collected during an experiment in which nitrate (a native electron acceptor) was injected into a perennially suboxic aquifer in Rifle (CO). Transcriptional data indicated that just two groups of chemolithoautotrophic bacteria accounted for a very large portion (~80%) of overall community gene expression: (1) members of the Fe(II)-oxidizing Gallionellaceae family and (2) strains of the S-oxidizing species, Sulfurimonas denitrificans. Metabolic lifestyles for Gallionellaceae strains that were novel compared to cultivated representatives included nitrate-dependent Fe(II) oxidation and S oxidation. Evidence for these metabolisms included highly correlated temporal expression in binned data of nitrate reductase (e.g., narGHI) genes (which have never been reported in Gallionellaceae genomes) and Fe(II) oxidation genes (e.g., mtoA) or S oxidation genes (e.g., dsrE, aprA). Of the two most active strains of S. denitrificans, only one showed strong expression of S oxidation genes, whereas the other was apparently using an unexpected (as-yet unidentified) primary electron donor. Transcriptional data added considerable interpretive value to this study, as (1) metagenomic data would not have highlighted these organisms, which had a disproportionately large role in community metabolism relative to their populations, and (2) co-expression of coupled pathway genes could not be predicted based solely on metagenomic data.

  18. An Unexpected Gas-Phase Binding Motif for Metal Dication Complexation with Peptides: Irmpd Spectroscopic Structure Determination

    NASA Astrophysics Data System (ADS)

    Dunbar, Robert C.; Steill, Jeffrey; Polfer, Nicolas; Berden, Giel; Oomens, Jos

    2011-06-01

    The favorable orientation of the amide linkage and the aromatic side chain of N-terminal Phe or Trp leads to several favorable motifs for metal ion binding to dipeptides, having distinct characteristics in the IR spectrum. Infrared multiple photon photodissociation spectroscopy using the FELIX free electron laser has enabled clear resolution of these isomeric forms. The spectral patterns of complexes of small dications (Mg2+, Ni2+ and Co2+) reveal an unexpected new isomeric form, in which the metal ion displaces the amide hydrogen, forming a metal-nitrogen bond with covalent character which is unprecedented in such gas-phase complexes. Spectra of the ions were acquired by irradiating the cell of the Fourier-transform ion cyclotron resonance mass spectrometer with infrared light from the FELIX laser at wavelengths in the approximate range 500 to 1900 Cm-1.

  19. High-throughput sequencing-based analysis of endogenetic fungal communities inhabiting the Chinese Cordyceps reveals unexpectedly high fungal diversity.

    PubMed

    Xia, Fei; Chen, Xin; Guo, Meng-Yuan; Bai, Xiao-Hui; Liu, Yan; Shen, Guang-Rong; Li, Yu-Ling; Lin, Juan; Zhou, Xuan-Wei

    2016-01-01

    Chinese Cordyceps, known in Chinese as "DongChong XiaCao", is a parasitic complex of a fungus (Ophiocordyceps sinensis) and a caterpillar. The current study explored the endogenetic fungal communities inhabiting Chinese Cordyceps. Samples were collected from five different geographical regions of Qinghai and Tibet, and the nuclear ribosomal internal transcribed spacer-1 sequences from each sample were obtained using Illumina high-throughput sequencing. The results showed that Ascomycota was the dominant fungal phylum in Chinese Cordyceps and its soil microhabitat from different sampling regions. Among the Ascomycota, 65 genera were identified, and the abundant operational taxonomic units showed the strongest sequence similarity to Ophiocordyceps, Verticillium, Pseudallescheria, Candida and Ilyonectria Not surprisingly, the genus Ophiocordyceps was the largest among the fungal communities identified in the fruiting bodies and external mycelial cortices of Chinese Cordyceps. In addition, fungal communities in the soil microhabitats were clustered separately from the external mycelial cortices and fruiting bodies of Chinese Cordyceps from different sampling regions. There was no significant structural difference in the fungal communities between the fruiting bodies and external mycelial cortices of Chinese Cordyceps. This study revealed an unexpectedly high diversity of fungal communities inhabiting the Chinese Cordyceps and its microhabitats. PMID:27625176

  20. Unexpected Pregnancy Revealed on 18F-NaF PET/CT.

    PubMed

    Shao, Fuqiang; Chen, Yue; Huang, Zhanwen; Cai, Liang; Zhang, Yin

    2016-04-01

    A 48-year-old illiterate woman who is congenitally deaf and mute underwent F-NaF PET/CT study to evaluate bone metastases from newly diagnosed breast cancer. Unexpectedly, a fetus in early second trimester was noted on CT images. In addition, subtle F-NaF uptake by the fetus could also be observed. PMID:26359575

  1. Metagenomic investigation of the geologically unique Hellenic Volcanic Arc reveals a distinctive ecosystem with unexpected physiology.

    PubMed

    Oulas, Anastasis; Polymenakou, Paraskevi N; Seshadri, Rekha; Tripp, H James; Mandalakis, Manolis; Paez-Espino, A David; Pati, Amrita; Chain, Patrick; Nomikou, Paraskevi; Carey, Steven; Kilias, Stephanos; Christakis, Christos; Kotoulas, Georgios; Magoulas, Antonios; Ivanova, Natalia N; Kyrpides, Nikos C

    2016-04-01

    Hydrothermal vents represent a deep, hot, aphotic biosphere where chemosynthetic primary producers, fuelled by chemicals from Earth's subsurface, form the basis of life. In this study, we examined microbial mats from two distinct volcanic sites within the Hellenic Volcanic Arc (HVA). The HVA is geologically and ecologically unique, with reported emissions of CO2 -saturated fluids at temperatures up to 220°C and a notable absence of macrofauna. Metagenomic data reveals highly complex prokaryotic communities composed of chemolithoautotrophs, some methanotrophs, and to our surprise, heterotrophs capable of anaerobic degradation of aromatic hydrocarbons. Our data suggest that aromatic hydrocarbons may indeed be a significant source of carbon in these sites, and instigate additional research into the nature and origin of these compounds in the HVA. Novel physiology was assigned to several uncultured prokaryotic lineages; most notably, a SAR406 representative is attributed with a role in anaerobic hydrocarbon degradation. This dataset, the largest to date from submarine volcanic ecosystems, constitutes a significant resource of novel genes and pathways with potential biotechnological applications. PMID:26487573

  2. In situ TEM straining of nanograined free-standing thin films reveals various unexpected deformation mechanisms.

    SciTech Connect

    Follstaedt, David Martin; Knapp, James Arthur; Clark, Blythe G.; Hattar, Khalid M.; Robertson, Ian M.

    2010-04-01

    In-situ transmission electron microscopy (TEM) straining experiments provide direct detailed observation of the deformation and failure mechanisms active at a length scale relevant to nanomaterials. This presentation will detail continued investigations into the active mechanisms governing high purity nanograined pulsed-laser deposited (PLD) nickel, as well as recent work into dislocation-particle interactions in nanostructured PLD aluminum-alumina alloys. Straining experiments performed on nanograined PLD free-standing nanograined Ni films with an engineered grain size distribution revealed that the addition of ductility with limited decrease in strength, reported in such metals, can be attributed to the simultaneous activity of three deformation mechanisms in front of the crack tip. At the crack tip, a grain agglomeration mechanism occurs where several nanograins appear to rotate, resulting in a very thin, larger grain immediately prior to failure. In the classical plastic zone in front of the crack tip, a multitude of mechanisms were found to operate in the larger grains including: dislocation pile-up, twinning, and stress-assisted grain growth. The region outside of the plastic zone showed signs of elasticity with limited indications of dislocation activity. The insight gained from in-situ TEM straining experiments of nanograined PLD Ni provides feedback for models of the deformation and failure in nanograined FCC metals, and suggests a greater complexity in the active mechanisms. The investigation into the deformation and failure mechanisms of FCC metals via in-situ TEM straining experiments has been expanded to the effect of hard particles on the active mechanisms in nanograined aluminum with alumina particles. The microstructures investigated were developed with varying composition, grain size, and particle distribution via tailoring of the PLD conditions and subsequent annealing. In order to develop microstructures suitable for in-situ deformation testing

  3. Unexpected divergence and lack of divergence revealed in continental Asian Cyornis flycatchers (Aves: Muscicapidae).

    PubMed

    Zhang, Zhen; Wang, Xiaoyang; Huang, Yuan; Olsson, Urban; Martinez, Jonathan; Alström, Per; Lei, Fumin

    2016-01-01

    The flycatcher genus Cyornis (Aves: Muscicapidae) comprises 25 species with Oriental distributions. Their relationships are poorly known. We analyzed the phylogenetic relationships of 70 individuals from 12 species and several subspecies of Cyornis based on three mitochondrial genes and five nuclear introns, with special focus on Chinese and Vietnamese populations of the monotypic C. hainanus and polytypic C. rubeculoides. We found no support for inclusion of C. concretus in Cyornis. Deep divergences were observed among different subspecies of C. banyumas and C. rubeculoides. C. rubeculoides glaucicomans was also shown to have a highly distinctive song, and we propose that it is treated as a distinctive Chinese endemic species, C. glaucicomans. In contrast, the south Vietnamese C. rubeculoides klossi, which has a disjunct distribution from the other subspecies of C. rubeculoides, along with a recently discovered population in Guangdong Province (China) with several plumage features reminiscent of C. r. klossi, were indistinguishable in all loci analyzed from the phenotypically markedly different C. hainanus. More research is needed to elucidate the reasons for this unexpected pattern. PMID:26358612

  4. Unexpected hormonal activity of a catechol equine estrogen metabolite reveals reversible glutathione conjugation

    PubMed Central

    Peng, Kuan-Wei; Chang, Minsun; Wang, Yue-Ting; Wang, Zhican; Qin, Zhihui; Bolton, Judy L.; Thatcher, Gregory R. J.

    2010-01-01

    4-Hydroxyequilenin (4-OHEN) is a major phase I metabolite of the equine estrogens present in widely prescribed hormone replacement formulations. 4-OHEN is autoxidized to an electrophilic o-quinone that has been shown to redox cycle, generating ROS, and to covalently modify proteins and DNA and thus potentially to act as a chemical carcinogen. To establish the ability of 4-OHEN to act as a hormonal carcinogen at the estrogen receptor (ER), estrogen responsive gene expression and proliferation were studied in ER(+) breast cancer cells. Recruitment by 4-OHEN of ER to estrogen responsive elements (ERE) of DNA in MCF-7 cells was also studied and observed. 4-OHEN was a potent estrogen, with additional weak activity associated with binding to the arylhydrocarbon receptor (AhR). The potency of 4-OHEN towards classical ERα mediated activity was unexpected given the reported rapid autoxidation and trapping of the resultant quinone by GSH. Addition of thiols to cell cultures did not attenuate the estrogenic activity of 4-OHEN and pre-formed thiol conjugates added to cell incubations only marginally reduced ERE-luciferase induction. On reaction of the 4OHEN-GSH conjugate with NADPH, 4-OHEN was observed to be regenerated at a rate dependent upon NADPH concentration, indicating that intracellular non-enzymatic and enzymatic regeneration of 4-OHEN accounts for the observed estrogenic activity of 4-OHEN. 4-OHEN is therefore capable of inducing chemical and hormonal pathways that may contribute to estrogen-dependent carcinogenesis, and trapping by cellular thiols does not provide a mechanism of termination of these pathways. PMID:20540524

  5. Unexpected technological heterogeneity in northern Arabia indicates complex Late Pleistocene demography at the gateway to Asia.

    PubMed

    Scerri, Eleanor M L; Groucutt, Huw S; Jennings, Richard P; Petraglia, Michael D

    2014-10-01

    The role and significance of the Arabian Peninsula in modern human dispersals out of Africa is currently contentious. While qualitative observations of similarities between Arabian Middle Palaeolithic and African Middle Stone Age (MSA) assemblages have been made, these inferences remain untested and often situated within overly broad dichotomies (e.g., 'Africa' versus the 'Levant'), which distort concepts of geographic scale and subsume local variability. Here, we quantitatively test the hypothesis that assemblages from Jubbah, in the Nefud Desert of northern Saudi Arabia are similar to MSA industries from northeast Africa. Based on the quantitative analysis of a suite of metric and morphological data describing lithic reduction sequences, our results show that early and late core reduction at Jubbah is distinct from equivalent northeast African strategies, perhaps as a result of raw material factors. However, specific techniques of core shaping, preparation and preferential flake production at Jubbah draw from a number of methods also present in the northeast African MSA. While two Jubbah lithic assemblages (JKF-1 and JKF-12) display both similarities and differences with the northeast African assemblages, a third locality (JSM-1) was significantly different to both the other Arabian and African assemblages, indicating an unexpected diversity of assemblages in the Jubbah basin during Marine Isotope Stage 5 (MIS 5, ∼125-70,000 years ago, or ka). Along with evidence from southern Arabia and the Levant, our results add quantitative support to arguments that MIS 5 hominin demography at the interface between Africa and Asia was complex. PMID:25110207

  6. Unexpected Diversity and Complexity of the Guerrero Negro Hypersaline Microbial Mat

    PubMed Central

    Ley, Ruth E.; Harris, J. Kirk; Wilcox, Joshua; Spear, John R.; Miller, Scott R.; Bebout, Brad M.; Maresca, Julia A.; Bryant, Donald A.; Sogin, Mitchell L.; Pace, Norman R.

    2006-01-01

    We applied nucleic acid-based molecular methods, combined with estimates of biomass (ATP), pigments, and microelectrode measurements of chemical gradients, to map microbial diversity vertically on a millimeter scale in a hypersaline microbial mat from Guerrero Negro, Baja California Sur, Mexico. To identify the constituents of the mat, small-subunit rRNA genes were amplified by PCR from community genomic DNA extracted from layers, cloned, and sequenced. Bacteria dominated the mat and displayed unexpected and unprecedented diversity. The majority (1,336) of the 1,586 bacterial 16S rRNA sequences generated were unique, representing 752 species (≥97% rRNA sequence identity) in 42 of the main bacterial phyla, including 15 novel candidate phyla. The diversity of the mat samples differentiated according to the chemical milieu defined by concentrations of O2 and H2S. Bacteria of the phylum Chloroflexi formed the majority of the biomass by percentage of bulk rRNA and of clones in rRNA gene libraries. This result contradicts the general belief that cyanobacteria dominate these communities. Although cyanobacteria constituted a large fraction of the biomass in the upper few millimeters (>80% of the total rRNA and photosynthetic pigments), Chloroflexi sequences were conspicuous throughout the mat. Filamentous Chloroflexi bacteria were identified by fluorescence in situ hybridization within the polysaccharide sheaths of the prominent cyanobacterium Microcoleus chthonoplastes, in addition to free living in the mat. The biological complexity of the mat far exceeds that observed in other polysaccharide-rich microbial ecosystems, such as the human and mouse distal guts, and suggests that positive feedbacks exist between chemical complexity and biological diversity. PMID:16672518

  7. Kinetics of tRNA(Pyl) -mediated amber suppression in Escherichia coli translation reveals unexpected limiting steps and competing reactions.

    PubMed

    Wang, Jinfan; Kwiatkowski, Marek; Forster, Anthony C

    2016-07-01

    The utility of ribosomal incorporation of unnatural amino acids (AAs) in vivo is generally restricted by low efficiencies, even with the most widely used suppressor tRNA(Pyl) . Because of the difficulties of studying incorporation in vivo, almost nothing is known about the limiting steps after tRNA charging. Here, we measured the kinetics of all subsequent steps using a purified Escherichia coli translation system. Dipeptide formation from initiator fMet-tRNA(fMet) and tRNA(Pyl) charged with allylglycine or methylserine displayed unexpectedly sluggish biphasic kinetics, ∼30-fold slower than for native substrates. The amplitude of the fast phases increased with increasing EF-Tu concentration, allowing measurement of Kd values of EF-Tu binding, both of which were ∼25-fold weaker than normal. However, binding could be increased ∼30-fold by lowering temperature. The fast phase rates were limited by the surprisingly ∼10-fold less efficient binding of EF-Tu:GTP:AA-tRNA(Pyl) ternary complex to the ribosomes, not GTP hydrolysis or peptide bond formation. Furthermore, processivity was unexpectedly impaired as ∼40% of the dipeptidyl-tRNA(Pyl) could not be elongated to tripeptide. Dipeptide formation was slow enough that termination due to misreading the UAG codon by non-cognate RF2 became very significant. This new understanding provides a framework for improving unnatural AA incorporation by amber suppression. Biotechnol. Bioeng. 2016;113: 1552-1559. © 2015 Wiley Periodicals, Inc. PMID:26705134

  8. Deep phenotyping of 89 xeroderma pigmentosum patients reveals unexpected heterogeneity dependent on the precise molecular defect

    PubMed Central

    Fassihi, Hiva; Sethi, Mieran; Fawcett, Heather; Wing, Jonathan; Chandler, Natalie; Mohammed, Shehla; Craythorne, Emma; Morley, Ana M. S.; Lim, Rongxuan; Turner, Sally; Henshaw, Tanya; Garrood, Isabel; Giunti, Paola; Hedderly, Tammy; Abiona, Adesoji; Naik, Harsha; Harrop, Gemma; McGibbon, David; Jaspers, Nicolaas G. J.; Botta, Elena; Nardo, Tiziana; Stefanini, Miria; Young, Antony R.; Sarkany, Robert P. E.; Lehmann, Alan R.

    2016-01-01

    Xeroderma pigmentosum (XP) is a rare DNA repair disorder characterized by increased susceptibility to UV radiation (UVR)-induced skin pigmentation, skin cancers, ocular surface disease, and, in some patients, sunburn and neurological degeneration. Genetically, it is assigned to eight complementation groups (XP-A to -G and variant). For the last 5 y, the UK national multidisciplinary XP service has provided follow-up for 89 XP patients, representing most of the XP patients in the United Kingdom. Causative mutations, DNA repair levels, and more than 60 clinical variables relating to dermatology, ophthalmology, and neurology have been measured, using scoring systems to categorize disease severity. This deep phenotyping has revealed unanticipated heterogeneity of clinical features, between and within complementation groups. Skin cancer is most common in XP-C, XP-E, and XP-V patients, previously considered to be the milder groups based on cellular analyses. These patients have normal sunburn reactions and are therefore diagnosed later and are less likely to adhere to UVR protection. XP-C patients are specifically hypersensitive to ocular damage, and XP-F and XP-G patients appear to be much less susceptible to skin cancer than other XP groups. Within XP groups, different mutations confer susceptibility or resistance to neurological damage. Our findings on this large cohort of XP patients under long-term follow-up reveal that XP is more heterogeneous than has previously been appreciated. Our data now enable provision of personalized prognostic information and management advice for each XP patient, as well as providing new insights into the functions of the XP proteins. PMID:26884178

  9. Deep phenotyping of 89 xeroderma pigmentosum patients reveals unexpected heterogeneity dependent on the precise molecular defect.

    PubMed

    Fassihi, Hiva; Sethi, Mieran; Fawcett, Heather; Wing, Jonathan; Chandler, Natalie; Mohammed, Shehla; Craythorne, Emma; Morley, Ana M S; Lim, Rongxuan; Turner, Sally; Henshaw, Tanya; Garrood, Isabel; Giunti, Paola; Hedderly, Tammy; Abiona, Adesoji; Naik, Harsha; Harrop, Gemma; McGibbon, David; Jaspers, Nicolaas G J; Botta, Elena; Nardo, Tiziana; Stefanini, Miria; Young, Antony R; Sarkany, Robert P E; Lehmann, Alan R

    2016-03-01

    Xeroderma pigmentosum (XP) is a rare DNA repair disorder characterized by increased susceptibility to UV radiation (UVR)-induced skin pigmentation, skin cancers, ocular surface disease, and, in some patients, sunburn and neurological degeneration. Genetically, it is assigned to eight complementation groups (XP-A to -G and variant). For the last 5 y, the UK national multidisciplinary XP service has provided follow-up for 89 XP patients, representing most of the XP patients in the United Kingdom. Causative mutations, DNA repair levels, and more than 60 clinical variables relating to dermatology, ophthalmology, and neurology have been measured, using scoring systems to categorize disease severity. This deep phenotyping has revealed unanticipated heterogeneity of clinical features, between and within complementation groups. Skin cancer is most common in XP-C, XP-E, and XP-V patients, previously considered to be the milder groups based on cellular analyses. These patients have normal sunburn reactions and are therefore diagnosed later and are less likely to adhere to UVR protection. XP-C patients are specifically hypersensitive to ocular damage, and XP-F and XP-G patients appear to be much less susceptible to skin cancer than other XP groups. Within XP groups, different mutations confer susceptibility or resistance to neurological damage. Our findings on this large cohort of XP patients under long-term follow-up reveal that XP is more heterogeneous than has previously been appreciated. Our data now enable provision of personalized prognostic information and management advice for each XP patient, as well as providing new insights into the functions of the XP proteins. PMID:26884178

  10. Uranus' Southern Circulation Revealed by Voyager-2 Images: Asymmetric, Unique, Unexpected

    NASA Astrophysics Data System (ADS)

    Karkoschka, Erich

    2014-11-01

    The southern half of Uranus' southern hemisphere of Uranus has been exceptionally bland. Only a single discrete feature was detected in Voyager-2 images, and none has been seen in thousands of HST and ground-based images since. All other observed regions on Uranus and jovian planets have many features that defined circulation patterns of the jovian planets, but the circulation of Uranus south of -45 deg latitude has been unknown.We performed a reanalysis of Voyager images of Uranus that revealed dozens of discrete features instead of the single feature known before. We improved flatfielding, pad-pixel treatment, and nonlinearity correction. We greatly decreased noise by averaging up to 1600 images. The result is a rotational profile without major gaps.Uranus' high southern latitudes are exceptional in several aspects: 1) The rotational profile has sharp kinks while it is smooth elsewhere on the ice giants. This puts current ideas of a simple Hadley cell on each hemisphere into question. 2) The rotational profile has a large north-south asymmetry, an order of magnitude larger than elsewhere on the jovian planets. 3) Between -68 and -59 deg latitude, the rotational shear is some 30 times lower than at other latitudes. Here, winds speeds around 200 m/s are regular to the 0.1 m/s level. 4) The South Pole had a spot off center rotating 5 h faster than the interior, which has not been observed elsewhere on jovian planets. 5) Uranus revealed spirals winding around the whole planet more than once that indicate very regular meridional motions, to the 2 cm/s level. 6) The latitude at -84 deg was featureless even at a signal-to-noise ratio of 55,000, one of the blandest zones in nature.Some features show significant evolution within the 5-week observing period providing constraints on dynamics. Features also show distinct spectral characteristics in the 8-filter data set providing constraints on the physical nature of features and their altitude. We have the data to

  11. The structure of sperm Izumo1 reveals unexpected similarities with Plasmodium invasion proteins.

    PubMed

    Nishimura, Kaoru; Han, Ling; Bianchi, Enrica; Wright, Gavin J; de Sanctis, Daniele; Jovine, Luca

    2016-07-25

    Fertilization, the culminating event in sexual reproduction, occurs when haploid sperm and egg recognize each other and fuse to form a diploid zygote. In mammals this process critically depends on the interaction between Izumo1, a protein exposed on the equatorial segment of acrosome-reacted sperm, and the egg plasma-membrane-anchored receptor Juno [1,2]. The molecular mechanism triggering gamete fusion is unresolved because both Izumo1 and Juno lack sequence similarity to known membrane fusogens. Here we report the crystal structure of Izumo1, which reveals a membrane distal region composed of a four-helix bundle connected to a carboxy-terminal immunoglobulin (Ig)-like domain through a β-hairpin stabilized by disulfide bonds. Remarkably, different regions of Izumo1 display significant structural similarities to two proteins expressed by the invasive sporozoite stage of Plasmodium parasites: SPECT1, which is essential for host cell traversal and hepatocyte invasion [3]; and TRAP, which is necessary for gliding motility and invasion [4]. These observations suggest a link between the molecular mechanisms underlying host cell invasion by the malaria parasite and gamete membrane fusion at fertilization. PMID:27374339

  12. Common and unexpected findings in mummies from ancient Egypt and South America as revealed by CT.

    PubMed

    Jackowski, Christian; Bolliger, Stephan; Thali, Michael J

    2008-01-01

    Computed tomography (CT) has proved to be a valuable investigative tool for mummy research and is the method of choice for examining mummies. It allows for noninvasive insight, especially with virtual endoscopy, which reveals detailed information about the mummy's sex, age, constitution, injuries, health, and mummification techniques used. CT also supplies three-dimensional information about the scanned object. Mummification processes can be summarized as "artificial," when the procedure was performed on a body with the aim of preservation, or as "natural," when the body's natural environment resulted in preservation. The purpose of artificial mummification was to preserve that person's morphologic features by delaying or arresting the decay of the body. The ancient Egyptians are most famous for this. Their use of evisceration followed by desiccation with natron (a compound of sodium salts) to halt putrefaction and prevent rehydration was so effective that their embalmed bodies have survived for nearly 4500 years. First, the body was cleaned with a natron solution; then internal organs were removed through the cribriform plate and abdomen. The most important, and probably the most lengthy, phase was desiccation. After the body was dehydrated, the body cavities were rinsed and packed to restore the body's former shape. Finally, the body was wrapped. Animals were also mummified to provide food for the deceased, to accompany the deceased as pets, because they were seen as corporal manifestations of deities, and as votive offerings. Artificial mummification was performed on every continent, especially in South and Central America. PMID:18794321

  13. Morphology and Molecules Reveal Unexpected Cryptic Diversity in the Enigmatic Genus Sinobirma Bryk, 1944 (Lepidoptera: Saturniidae)

    PubMed Central

    Rougerie, Rodolphe; Naumann, Stefan; Nässig, Wolfgang A.

    2012-01-01

    The wild silkmoth genus Sinobirma Bryk, 1944 is a poorly known monotypic taxon from the eastern end of the Himalaya Range. It was convincingly proposed to be closely related to some members of an exclusively Afro-tropical group of Saturniidae, but its biogeographical and evolutionary history remains enigmatic. After examining recently collected material from Tibet, northern India, and northeastern Myanmar, we realized that this unique species, S. malaisei Bryk, 1944 only known so far from a few specimens and from a very restricted area near the border between north-eastern Myanmar and the Yunnan province of China, may in fact belong to a group of closely related cryptic species. In this work, we combined morphological comparative study, DNA barcoding, and the sequences of a nuclear marker (D2 expansion segment of the 28S rRNA gene) to unequivocally delimit three distinct species in the genus Sinobirma, of which two are described as new to science: S. myanmarensis sp. n. and S. bouyeri sp. n. An informative DNA barcode sequence was obtained from the female holotype of S. malaisei—collected in 1934—ensuring the proper assignation of this name to the newly collected and studied specimens. Our findings represent another example of the potential of coupling traditional taxonomy and DNA barcoding for revealing and solving difficult cases of cryptic diversity. This approach is now being generalized to the world fauna of Saturniidae, with the participation of most of the taxonomists studying these moths. PMID:23028478

  14. Morphology and molecules reveal unexpected cryptic diversity in the enigmatic genus Sinobirma Bryk, 1944 (Lepidoptera: Saturniidae).

    PubMed

    Rougerie, Rodolphe; Naumann, Stefan; Nässig, Wolfgang A

    2012-01-01

    The wild silkmoth genus Sinobirma Bryk, 1944 is a poorly known monotypic taxon from the eastern end of the Himalaya Range. It was convincingly proposed to be closely related to some members of an exclusively Afro-tropical group of Saturniidae, but its biogeographical and evolutionary history remains enigmatic. After examining recently collected material from Tibet, northern India, and northeastern Myanmar, we realized that this unique species, S. malaisei Bryk, 1944 only known so far from a few specimens and from a very restricted area near the border between north-eastern Myanmar and the Yunnan province of China, may in fact belong to a group of closely related cryptic species. In this work, we combined morphological comparative study, DNA barcoding, and the sequences of a nuclear marker (D2 expansion segment of the 28S rRNA gene) to unequivocally delimit three distinct species in the genus Sinobirma, of which two are described as new to science: S. myanmarensis sp. n. and S. bouyeri sp. n. An informative DNA barcode sequence was obtained from the female holotype of S. malaisei--collected in 1934--ensuring the proper assignation of this name to the newly collected and studied specimens. Our findings represent another example of the potential of coupling traditional taxonomy and DNA barcoding for revealing and solving difficult cases of cryptic diversity. This approach is now being generalized to the world fauna of Saturniidae, with the participation of most of the taxonomists studying these moths. PMID:23028478

  15. Integrated Analyses Resolve Conflicts over Squamate Reptile Phylogeny and Reveal Unexpected Placements for Fossil Taxa

    PubMed Central

    Reeder, Tod W.; Townsend, Ted M.; Mulcahy, Daniel G.; Noonan, Brice P.; Wood, Perry L.; Sites, Jack W.; Wiens, John J.

    2015-01-01

    Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have become increasingly controversial. Squamates include >9000 species, making them the second largest group of terrestrial vertebrates. They are important medicinally and as model systems for ecological and evolutionary research. However, studies of squamate biology are hindered by uncertainty over their relationships, and some consider squamate phylogeny unresolved, given recent conflicts between molecular and morphological results. To resolve these conflicts, we expand existing morphological and molecular datasets for squamates (691 morphological characters and 46 genes, for 161 living and 49 fossil taxa, including a new set of 81 morphological characters and adding two genes from published studies) and perform integrated analyses. Our results resolve higher-level relationships as indicated by molecular analyses, and reveal hidden morphological support for the molecular hypothesis (but not vice-versa). Furthermore, we find that integrating molecular, morphological, and paleontological data leads to surprising placements for two major fossil clades (Mosasauria and Polyglyphanodontia). These results further demonstrate the importance of combining fossil and molecular information, and the potential problems of estimating the placement of fossil taxa from morphological data alone. Thus, our results caution against estimating fossil relationships without considering relevant molecular data, and against placing fossils into molecular trees (e.g. for dating analyses) without considering the possible impact of molecular data on their placement. PMID:25803280

  16. Proteomic Investigation of Aphid Honeydew Reveals an Unexpected Diversity of Proteins

    PubMed Central

    Haubruge, Eric; Hance, Thierry; Thonart, Philippe; De Pauw, Edwin; Francis, Frédéric

    2013-01-01

    Aphids feed on the phloem sap of plants, and are the most common honeydew-producing insects. While aphid honeydew is primarily considered to comprise sugars and amino acids, its protein diversity has yet to be documented. Here, we report on the investigation of the honeydew proteome from the pea aphid Acyrthosiphon pisum. Using a two-Dimensional Differential in-Gel Electrophoresis (2D-Dige) approach, more than 140 spots were isolated, demonstrating that aphid honeydew also represents a diverse source of proteins. About 66% of the isolated spots were identified through mass spectrometry analysis, revealing that the protein diversity of aphid honeydew originates from several organisms (i.e. the host aphid and its microbiota, including endosymbiotic bacteria and gut flora). Interestingly, our experiments also allowed to identify some proteins like chaperonin, GroEL and Dnak chaperones, elongation factor Tu (EF-Tu), and flagellin that might act as mediators in the plant-aphid interaction. In addition to providing the first aphid honeydew proteome analysis, we propose to reconsider the importance of this substance, mainly acknowledged to be a waste product, from the aphid ecology perspective. PMID:24086359

  17. Integrated analyses resolve conflicts over squamate reptile phylogeny and reveal unexpected placements for fossil taxa.

    PubMed

    Reeder, Tod W; Townsend, Ted M; Mulcahy, Daniel G; Noonan, Brice P; Wood, Perry L; Sites, Jack W; Wiens, John J

    2015-01-01

    Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have become increasingly controversial. Squamates include >9000 species, making them the second largest group of terrestrial vertebrates. They are important medicinally and as model systems for ecological and evolutionary research. However, studies of squamate biology are hindered by uncertainty over their relationships, and some consider squamate phylogeny unresolved, given recent conflicts between molecular and morphological results. To resolve these conflicts, we expand existing morphological and molecular datasets for squamates (691 morphological characters and 46 genes, for 161 living and 49 fossil taxa, including a new set of 81 morphological characters and adding two genes from published studies) and perform integrated analyses. Our results resolve higher-level relationships as indicated by molecular analyses, and reveal hidden morphological support for the molecular hypothesis (but not vice-versa). Furthermore, we find that integrating molecular, morphological, and paleontological data leads to surprising placements for two major fossil clades (Mosasauria and Polyglyphanodontia). These results further demonstrate the importance of combining fossil and molecular information, and the potential problems of estimating the placement of fossil taxa from morphological data alone. Thus, our results caution against estimating fossil relationships without considering relevant molecular data, and against placing fossils into molecular trees (e.g. for dating analyses) without considering the possible impact of molecular data on their placement. PMID:25803280

  18. The transcriptome of Euglena gracilis reveals unexpected metabolic capabilities for carbohydrate and natural product biochemistry.

    PubMed

    O'Neill, Ellis C; Trick, Martin; Hill, Lionel; Rejzek, Martin; Dusi, Renata G; Hamilton, Chris J; Zimba, Paul V; Henrissat, Bernard; Field, Robert A

    2015-10-01

    Euglena gracilis is a highly complex alga belonging to the green plant line that shows characteristics of both plants and animals, while in evolutionary terms it is most closely related to the protozoan parasites Trypanosoma and Leishmania. This well-studied organism has long been known as a rich source of vitamins A, C and E, as well as amino acids that are essential for the human diet. Here we present de novo transcriptome sequencing and preliminary analysis, providing a basis for the molecular and functional genomics studies that will be required to direct metabolic engineering efforts aimed at enhancing the quality and quantity of high value products from E. gracilis. The transcriptome contains over 30,000 protein-encoding genes, supporting metabolic pathways for lipids, amino acids, carbohydrates and vitamins, along with capabilities for polyketide and non-ribosomal peptide biosynthesis. The metabolic and environmental robustness of Euglena is supported by a substantial capacity for responding to biotic and abiotic stress: it has the capacity to deploy three separate pathways for vitamin C (ascorbate) production, as well as producing vitamin E (α-tocopherol) and, in addition to glutathione, the redox-active thiols nor-trypanothione and ovothiol. PMID:26289754

  19. The Structure of Plasmodium falciparum Blood-Stage 6-Cys Protein Pf41 Reveals an Unexpected Intra-Domain Insertion Required for Pf12 Coordination.

    PubMed

    Parker, Michelle L; Peng, Fangni; Boulanger, Martin J

    2015-01-01

    Plasmodium falciparum is an apicomplexan parasite and the etiological agent of severe human malaria. The complex P. falciparum life cycle is supported by a diverse repertoire of surface proteins including the family of 6-Cys s48/45 antigens. Of these, Pf41 is localized to the surface of the blood-stage merozoite through its interaction with the glycophosphatidylinositol-anchored Pf12. Our recent structural characterization of Pf12 revealed two juxtaposed 6-Cys domains (D1 and D2). Pf41, however, contains an additional segment of 120 residues predicted to form a large spacer separating its two 6-Cys domains. To gain insight into the assembly mechanism and overall architecture of the Pf12-Pf41 complex, we first determined the 2.45 Å resolution crystal structure of Pf41 using zinc single-wavelength anomalous dispersion. Structural analysis revealed an unexpected domain organization where the Pf41 6-Cys domains are, in fact, intimately associated and the additional residues instead map predominately to an inserted domain-like region (ID) located between two β-strands in D1. Notably, the ID is largely proteolyzed in the final structure suggesting inherent flexibility. To assess the contribution of the ID to complex formation, we engineered a form of Pf41 where the ID was replaced by a short glycine-serine linker and showed by isothermal titration calorimetry that binding to Pf12 was abrogated. Finally, protease protection assays showed that the proteolytic susceptibility of the ID was significantly reduced in the complex, consistent with the Pf41 ID directly engaging Pf12. Collectively, these data establish the architectural organization of Pf41 and define an essential role for the Pf41 ID in promoting assembly of the Pf12-Pf41 heterodimeric complex. PMID:26414347

  20. Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ.

    PubMed

    Li, Yi; Chirgadze, Dimitri Y; Bolanos-Garcia, Victor M; Sibanda, Bancinyane L; Davies, Owen R; Ahnesorg, Peter; Jackson, Stephen P; Blundell, Tom L

    2008-01-01

    The recently characterised 299-residue human XLF/Cernunnos protein plays a crucial role in DNA repair by non-homologous end joining (NHEJ) and interacts with the XRCC4-DNA Ligase IV complex. Here, we report the crystal structure of the XLF (1-233) homodimer at 2.3 A resolution, confirming the predicted structural similarity to XRCC4. The XLF coiled-coil, however, is shorter than that of XRCC4 and undergoes an unexpected reverse in direction giving rise to a short distorted four helical bundle and a C-terminal helical structure wedged between the coiled-coil and head domain. The existence of a dimer as the major species is confirmed by size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering and other biophysical methods. We show that the XLF structure is not easily compatible with a proposed XRCC4:XLF heterodimer. However, we demonstrate interactions between dimers of XLF and XRCC4 by surface plasmon resonance and analyse these in terms of surface properties, amino-acid conservation and mutations in immunodeficient patients. Our data are most consistent with head-to-head interactions in a 2:2:1 XRCC4:XLF:Ligase IV complex. PMID:18046455

  1. Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis

    PubMed Central

    2012-01-01

    Background Filamentous fungi are potent biomass degraders due to their ability to thrive in ligno(hemi)cellulose-rich environments. During the last decade, fungal genome sequencing initiatives have yielded abundant information on the genes that are putatively involved in lignocellulose degradation. At present, additional experimental studies are essential to provide insights into the fungal secreted enzymatic pools involved in lignocellulose degradation. Results In this study, we performed a wide analysis of 20 filamentous fungi for which genomic data are available to investigate their biomass-hydrolysis potential. A comparison of fungal genomes and secretomes using enzyme activity profiling revealed discrepancies in carbohydrate active enzymes (CAZymes) sets dedicated to plant cell wall. Investigation of the contribution made by each secretome to the saccharification of wheat straw demonstrated that most of them individually supplemented the industrial Trichoderma reesei CL847 enzymatic cocktail. Unexpectedly, the most striking effect was obtained with the phytopathogen Ustilago maydis that improved the release of total sugars by 57% and of glucose by 22%. Proteomic analyses of the best-performing secretomes indicated a specific enzymatic mechanism of U. maydis that is likely to involve oxido-reductases and hemicellulases. Conclusion This study provides insight into the lignocellulose-degradation mechanisms by filamentous fungi and allows for the identification of a number of enzymes that are potentially useful to further improve the industrial lignocellulose bioconversion process. PMID:22300648

  2. Kinetic Analysis as a Tool to Distinguish Pathway Complexity in Molecular Assembly: An Unexpected Outcome of Structures in Competition.

    PubMed

    van der Zwaag, Daan; Pieters, Pascal A; Korevaar, Peter A; Markvoort, Albert J; Spiering, A J H; de Greef, Tom F A; Meijer, E W

    2015-10-01

    While the sensitive dependence of the functional characteristics of self-assembled nanofibers on the molecular structure of their building blocks is well-known, the crucial influence of the dynamics of the assembly process is often overlooked. For natural protein-based fibrils, various aggregation mechanisms have been demonstrated, from simple primary nucleation to secondary nucleation and off-pathway aggregation. Similar pathway complexity has recently been described in synthetic supramolecular polymers and has been shown to be intimately linked to their morphology. We outline a general method to investigate the consequences of the presence of multiple assembly pathways, and show how kinetic analysis can be used to distinguish different assembly mechanisms. We illustrate our combined experimental and theoretical approach by studying the aggregation of chiral bipyridine-extended 1,3,5-benzenetricarboxamides (BiPy-1) in n-butanol as a model system. Our workflow consists of nonlinear least-squares analysis of steady-state spectroscopic measurements, which cannot provide conclusive mechanistic information but yields the equilibrium constants of the self-assembly process as constraints for subsequent kinetic analysis. Furthermore, kinetic nucleation-elongation models based on one and two competing pathways are used to interpret time-dependent spectroscopic measurements acquired using stop-flow and temperature-jump methods. Thus, we reveal that the sharp transition observed in the aggregation process of BiPy-1 cannot be explained by a single cooperative pathway, but can be described by a competitive two-pathway mechanism. This work provides a general tool for analyzing supramolecular polymerizations and establishing energetic landscapes, leading to mechanistic insights that at first sight may seem unexpected and counterintuitive. PMID:26354151

  3. Metatranscriptomic analysis of a high-sulfide aquatic spring reveals insights into sulfur cycling and unexpected aerobic metabolism.

    PubMed

    Spain, Anne M; Elshahed, Mostafa S; Najar, Fares Z; Krumholz, Lee R

    2015-01-01

    Zodletone spring is a sulfide-rich spring in southwestern Oklahoma characterized by shallow, microoxic, light-exposed spring water overlaying anoxic sediments. Previously, culture-independent 16S rRNA gene based diversity surveys have revealed that Zodletone spring source sediments harbor a highly diverse microbial community, with multiple lineages putatively involved in various sulfur-cycling processes. Here, we conducted a metatranscriptomic survey of microbial populations in Zodletone spring source sediments to characterize the relative prevalence and importance of putative phototrophic, chemolithotrophic, and heterotrophic microorganisms in the sulfur cycle, the identity of lineages actively involved in various sulfur cycling processes, and the interaction between sulfur cycling and other geochemical processes at the spring source. Sediment samples at the spring's source were taken at three different times within a 24-h period for geochemical analyses and RNA sequencing. In depth mining of datasets for sulfur cycling transcripts revealed major sulfur cycling pathways and taxa involved, including an unexpected potential role of Actinobacteria in sulfide oxidation and thiosulfate transformation. Surprisingly, transcripts coding for the cyanobacterial Photosystem II D1 protein, methane monooxygenase, and terminal cytochrome oxidases were encountered, indicating that genes for oxygen production and aerobic modes of metabolism are actively being transcribed, despite below-detectable levels (<1 µM) of oxygen in source sediment. Results highlight transcripts involved in sulfur, methane, and oxygen cycles, propose that oxygenic photosynthesis could support aerobic methane and sulfide oxidation in anoxic sediments exposed to sunlight, and provide a viewpoint of microbial metabolic lifestyles under conditions similar to those seen during late Archaean and Proterozoic eons. PMID:26417542

  4. Metatranscriptomic analysis of a high-sulfide aquatic spring reveals insights into sulfur cycling and unexpected aerobic metabolism

    PubMed Central

    Elshahed, Mostafa S.; Najar, Fares Z.; Krumholz, Lee R.

    2015-01-01

    Zodletone spring is a sulfide-rich spring in southwestern Oklahoma characterized by shallow, microoxic, light-exposed spring water overlaying anoxic sediments. Previously, culture-independent 16S rRNA gene based diversity surveys have revealed that Zodletone spring source sediments harbor a highly diverse microbial community, with multiple lineages putatively involved in various sulfur-cycling processes. Here, we conducted a metatranscriptomic survey of microbial populations in Zodletone spring source sediments to characterize the relative prevalence and importance of putative phototrophic, chemolithotrophic, and heterotrophic microorganisms in the sulfur cycle, the identity of lineages actively involved in various sulfur cycling processes, and the interaction between sulfur cycling and other geochemical processes at the spring source. Sediment samples at the spring’s source were taken at three different times within a 24-h period for geochemical analyses and RNA sequencing. In depth mining of datasets for sulfur cycling transcripts revealed major sulfur cycling pathways and taxa involved, including an unexpected potential role of Actinobacteria in sulfide oxidation and thiosulfate transformation. Surprisingly, transcripts coding for the cyanobacterial Photosystem II D1 protein, methane monooxygenase, and terminal cytochrome oxidases were encountered, indicating that genes for oxygen production and aerobic modes of metabolism are actively being transcribed, despite below-detectable levels (<1 µM) of oxygen in source sediment. Results highlight transcripts involved in sulfur, methane, and oxygen cycles, propose that oxygenic photosynthesis could support aerobic methane and sulfide oxidation in anoxic sediments exposed to sunlight, and provide a viewpoint of microbial metabolic lifestyles under conditions similar to those seen during late Archaean and Proterozoic eons. PMID:26417542

  5. Extending motifs in lithiocuprate chemistry: unexpected structural diversity in thiocyanate complexes.

    PubMed

    Peel, Andrew J; Hedidi, Madani; Bentabed-Ababsa, Ghenia; Roisnel, Thierry; Mongin, Florence; Wheatley, Andrew E H

    2016-04-14

    The new area of lithio(thiocyanato)cuprates has been developed. Using inexpensive, stable and safe CuSCN for their preparation, these complexes revealed Lipshutz-type dimeric motifs with solvent-dependent point group identities; planar, boat-shaped and chair shaped conformers are seen in the solid state. In solution, both Lipshutz-type and Gilman structures are clearly seen. Since the advent in 2007 of directed ortho cupration, effort has gone into understanding the structure-reactivity effects of amide ligand variation in and alkali metal salt abstraction from Lipshutz-type cuprates such as (TMP)2Cu(CN)Li2(THF) 1 (TMP = 2,2,6,6-tetramethylpiperidide). The replacement of CN(-) with SCN(-) is investigated presently as a means of improving the safety of lithium cuprates. The synthesis and solid state structural characterization of reference cuprate (TMP)2Cu(CN)Li2(THP) 8 (THP = tetrahydropyran) precedes that of the thiocyanate series (TMP)2Cu(SCN)Li2(L) (L = OEt29, THF 10, THP 11). For each of 9-11, preformed TMPLi was combined with CuSCN (2 : 1) in the presence of sub-stoichiometric Lewis base (0.5 eq. wrt Li). The avoidance of Lewis basic solvents incurs formation of the unsolvated Gilman cuprate (TMP)2CuLi 12, whilst multidimensional NMR spectroscopy has evidenced the abstraction of LiSCN from 9-11 in hydrocarbon solution and the in situ formation of Gilman reagents. The synthetic utility of 10 is established in the selective deprotometalation of chloropyridine substrates, including effecting transition metal-free homocoupling in 51-69% yield. PMID:26554572

  6. Unexpected Ancient Paralogs and an Evolutionary Model for the COPII Coat Complex

    PubMed Central

    Schlacht, Alexander; Dacks, Joel B.

    2015-01-01

    The coat protein complex II (COPII) is responsible for the transport of protein cargoes from the Endoplasmic Reticulum (ER) to the Golgi apparatus. COPII has been functionally characterized extensively in vivo in humans and yeast. This complex shares components with the nuclear pore complex and the Seh1-Associated (SEA) complex, inextricably linking its evolution with that of the nuclear pore and other protocoatomer domain-containing complexes. Importantly, this is one of the last coat complexes to be examined from a comparative genomic and phylogenetic perspective. We use homology searching of eight components across 74 eukaryotic genomes, followed by phylogenetic analyses, to assess both the distribution of the COPII components across eukaryote diversity and to assess its evolutionary history. We report that Sec12, but not Sed4 was present in the Last Eukaryotic Common Ancestor along with Sec16, Sar1, Sec13, Sec31, Sec23, and Sec24. We identify a previously undetected paralog of Sec23 that, at least, predates the archaeplastid clade. We also describe three Sec24 paralogs likely present in the Last Eukaryotic Common Ancestor, including one newly detected that was anciently present but lost from both opisthokonts and excavates. Altogether, we report previously undescribed complexity of the COPII coat in the ancient eukaryotic ancestor and speculate on models for the evolution, not only of the complex, but its relationship to other protocoatomer-derived complexes. PMID:25747251

  7. The synthesis and unexpected solution chemistry of thermochromic carborane-containing osmium half-sandwich complexes.

    PubMed

    Pitto-Barry, Anaïs; South, Amy; Rodger, Alison; Barry, Nicolas P E

    2016-01-28

    The functionalisation of the 16-electron complex [Os(η(6)-p-cymene)(1,2-dicarba-closo-dodecarborane-1,2-dithiolato)] (1) with a series of Lewis bases to give the 18-electron complexes of general formula [Os(η(6)-p-cymene)(1,2-dicarba-closo-dodecarborane-1,2-dithiolato)(L)] (L = pyridine (2), 4-dimethylaminopyridine (3), 4-cyanopyridine (4), 4-methoxypyridine (5), pyrazine (6), pyridazine (7), 4,4'-bipyridine (8) and triphenylphosphine (9)) is reported. All 18-electron complexes are in equilibrium in solution with the 16-electron precursor, and thermochromic properties are observed in some cases (2, 3, 5, 8, and 9). The binding constants and Gibbs free energies of the equilibria are determined using UV-visible titrations and their stabilities investigated. Synthetic routes for forcing the formation of the 18-electron species are proposed, and analytical methods to characterise the equilibria are described. PMID:26700880

  8. Mechanistic basis for unexpected bioavailability enhancement of polyelectrolyte complexes incorporating BCS class III drugs and carrageenans.

    PubMed

    Heinen, C; Reuss, S; Saaler-Reinhardt, S; Langguth, P

    2013-09-01

    The objective of this study was to investigate the potential of λ-carrageenan to work as an absorption modifying excipient in combination with formulations of BCS class 3 substances. Trospium chloride was used as a model BCS class 3 substance. Polyelectrolyte complexes of trospium and λ-carrageenan were produced by layer-by-layer complexation. A λ-carrageenan-containing formulation was administered either in capsules size 9 to rats by gavage or directly into ligated intestinal loops of rats. Exceptionally strong variations were observed in the plasma concentrations of the rats that received λ-carrageenan compared to the control group, but enhanced plasma concentrations were observed only in some of the rats. In vitro permeability studies were performed across Caco2-monolayers and across excised segments of rat jejunum in a modified Ussing chamber to learn more about the mechanism of absorption enhancement. The complex did not show any effect in Caco2-cells, but led to a major enhancement of permeability across excised segments in modified Ussing chambers. Carrageenan did not lead to alterations of tight junctions. The bioavailability enhancing effect thus was most likely due to an interaction of the polyelectrolyte-drug complex with the mucus, which provided an intimate contact between the drug and the absorbing surface. A similar effect was also achievable with other types of carrageenan and was also transferable to other compounds. In conclusion, λ-carrageenan-drug complexes show interesting excipient-drug-epithelium interactions - however, for full utilization of the permeation enhancing potential, an intimate and reproducible contact between absorbing epithelia and the complex is needed. PMID:23958316

  9. Unexpected formation of a novel pyridinium-containing catecholate ligand and its manganese(III) complex.

    PubMed

    Sheriff, Tippu S; Watkinson, Michael; Motevalli, Majid; Lesin, Jocelyne F

    2010-01-01

    Nucleophilic aromatic substitution of tetrachloro-o-benzoquinone by pyridine and reduction of the o-quinone to the catechol by hydroxylamine forms 1,2-dihydroxy-3,5,6-trichlorobenzene-4-pyridinium chloride. This compound reacts with manganese(II) acetate in air to form chlorobis(3,5,6-trichlorobenzene 4-pyridinium catecholate)manganese(III), which represents the first complex of this ligand class to be structurally characterized by X-ray diffraction; this complex is active in the catalytic reduction of dioxygen to hydrogen peroxide under ambient conditions and turnover frequencies (TOFs) >10,000 h(-1) can be obtained. PMID:20023930

  10. Bio-mimicking of Proline-Rich Motif Applied to Carbon Nanotube Reveals Unexpected Subtleties Underlying Nanoparticle Functionalization

    PubMed Central

    Zhang, Yuanzhao; Jimenez-Cruz, Camilo A.; Wang, Jian; Zhou, Bo; Yang, Zaixing; Zhou, Ruhong

    2014-01-01

    Here, we report computational studies of the SH3 protein domain interacting with various single-walled carbon nanotubes (SWCNT) either bare or functionalized by mimicking the proline-rich motif (PRM) ligand (PPPVPPRR) and compare it to the SH3-PRM complex binding. With prolines or a single arginine attached, the SWCNT gained slightly on specificity when compared with the bare control, whereas with multi-arginine systems the specificity dropped dramatically to our surprise. Although the electrostatic interaction provided by arginines is crucial in the recognition between PRM and SH3 domain, our results suggest that attaching multiple arginines to the SWCNT has a detrimental effect on the binding affinity. Detailed analysis of the MD trajectories found two main factors that modulate the specificity of the binding: the existence of competing acidic patches at the surface of SH3 that leads to “trapping and clamping” by the arginines, and the rigidity of the SWCNT introducing entropic penalties in the proper binding. Further investigation revealed that the same “clamping” phenomenon exits in the PRM-SH3 system, which has not been reported in previous literature. The competing effects between nanoparticle and its functionalization components revealed by our model system should be of value to current and future nanomedicine designs. PMID:25427563

  11. Bio-mimicking of proline-rich motif applied to carbon nanotube reveals unexpected subtleties underlying nanoparticle functionalization.

    PubMed

    Zhang, Yuanzhao; Jimenez-Cruz, Camilo A; Wang, Jian; Zhou, Bo; Yang, Zaixing; Zhou, Ruhong

    2014-01-01

    Here, we report computational studies of the SH3 protein domain interacting with various single-walled carbon nanotubes (SWCNT) either bare or functionalized by mimicking the proline-rich motif (PRM) ligand (PPPVPPRR) and compare it to the SH3-PRM complex binding. With prolines or a single arginine attached, the SWCNT gained slightly on specificity when compared with the bare control, whereas with multi-arginine systems the specificity dropped dramatically to our surprise. Although the electrostatic interaction provided by arginines is crucial in the recognition between PRM and SH3 domain, our results suggest that attaching multiple arginines to the SWCNT has a detrimental effect on the binding affinity. Detailed analysis of the MD trajectories found two main factors that modulate the specificity of the binding: the existence of competing acidic patches at the surface of SH3 that leads to "trapping and clamping" by the arginines, and the rigidity of the SWCNT introducing entropic penalties in the proper binding. Further investigation revealed that the same "clamping" phenomenon exits in the PRM-SH3 system, which has not been reported in previous literature. The competing effects between nanoparticle and its functionalization components revealed by our model system should be of value to current and future nanomedicine designs. PMID:25427563

  12. Micro-CT scan reveals an unexpected high-volume and interconnected pore network in a Cretaceous Sanagasta dinosaur eggshell.

    PubMed

    Hechenleitner, E Martín; Grellet-Tinner, Gerald; Foley, Matthew; Fiorelli, Lucas E; Thompson, Michael B

    2016-03-01

    The Cretaceous Sanagasta neosauropod nesting site (La Rioja, Argentina) was the first confirmed instance of extinct dinosaurs using geothermal-generated heat to incubate their eggs. The nesting strategy and hydrothermal activities at this site led to the conclusion that the surprisingly 7 mm thick-shelled eggs were adapted to harsh hydrothermal microenvironments. We used micro-CT scans in this study to obtain the first three-dimensional microcharacterization of these eggshells. Micro-CT-based analyses provide a robust assessment of gas conductance in fossil dinosaur eggshells with complex pore canal systems, allowing calculation, for the first time, of the shell conductance through its thickness. This novel approach suggests that the shell conductance could have risen during incubation to seven times more than previously estimated as the eggshell erodes. In addition, micro-CT observations reveal that the constant widening and branching of pore canals form a complex funnel-like pore canal system. Furthermore, the high density of pore canals and the presence of a lateral canal network in the shell reduce the risks of pore obstruction during the extended incubation of these eggs in a relatively highly humid and muddy nesting environment. PMID:27009182

  13. Highly asymmetric bromocyclization of tryptophol: unexpected accelerating effect of DABCO-derived bromine complex.

    PubMed

    Liu, Huan; Jiang, Guangde; Pan, Xixian; Wan, Xiaolong; Lai, Yisheng; Ma, Dawei; Xie, Weiqing

    2014-04-01

    Highly asymmetric bromocyclization of tryptophol by using chiral anionic phase-transfer catalyst and DABCO-derived brominating reagent is described. Optimization of the reaction conditions revealed that the reaction rate was accelerated together with improvement of enantioselectivity by addition of catalytic DABCO-derived brominating reagent. From tryptophol, 3-bromofuroindoline could be directly obtained in excellent enantioselectivities by employing this novel methodology. PMID:24666363

  14. Unexpected Outcomes of Thai Cassava Trade: A Case of Global Complexity and Local Unsustainability

    PubMed Central

    CURRAN, SARA R.; COOKE, ABIGAIL M.

    2014-01-01

    Tracing the Thai cassava (Manihot esculenta) trade network, between 1960 and 2000, offers a compelling example of global complexity at work. The emergence of Thailand’s dominance of world export markets caught the world by surprise. The opening up of a European market for cassava was supposed to be met by Brazilian and Indonesian producers. Instead, Thailand took over the market by 1975. Several factors facilitated this emergence including: entrepreneurial diasporic networks of Thai-Chinese traders, local political economy conditions in both Europe and Thailand, and ecological conditions in Thailand. These same factors also shaped the subsequent timing of the closing of the European market, the emergence of a new industry association, the creation of new cassava products, and the expansion to other markets. Furthermore, the dynamic nature of cassava market yielded equivocal outcomes for both Europe and Thai farmers. PMID:25328444

  15. Complex communities of small protists and unexpected occurrence of typical marine lineages in shallow freshwater systems

    PubMed Central

    Simon, Marianne; Jardillier, Ludwig; Deschamps, Philippe; Moreira, David; Restoux, Gwendal; Bertolino, Paola; López-García, Purificación

    2014-01-01

    Summary Although inland water bodies are more heterogeneous and sensitive to environmental variation than oceans, the diversity of small protists in these ecosystems is much less well-known. Some molecular surveys of lakes exist, but little information is available from smaller, shallower and often ephemeral freshwater systems, despite their global distribution and ecological importance. We carried out a comparative study based on massive pyrosequencing of amplified 18S rRNA gene fragments of protists in the 0.2-5 μm-size range in one brook and four shallow ponds located in the Natural Regional Park of the Chevreuse Valley, France. Our study revealed a wide diversity of small protists, with 812 stringently defined operational taxonomic units (OTUs) belonging to the recognized eukaryotic supergroups (SAR –Stramenopiles, Alveolata, Rhizaria–, Archaeplastida, Excavata, Amoebozoa, Opisthokonta) and to groups of unresolved phylogenetic position (Cryptophyta, Haptophyta, Centrohelida, Katablepharida, Telonemida, Apusozoa). Some OTUs represented deep-branching lineages (Cryptomycota, Aphelida, Colpodellida, Tremulida, clade-10 Cercozoa, HAP-1 Haptophyta). We identified several lineages previously thought to be marine including, in addition to MAST-2 and MAST-12, already detected in freshwater, MAST-3 and possibly MAST-6. Protist community structures were different in the five ecosystems. These differences did not correlate with geographical distances, but seemed to be influenced by environmental parameters. PMID:25115943

  16. Unexpected complexity of the Reef-Building Coral Acropora millepora transcription factor network

    PubMed Central

    2011-01-01

    Background Coral reefs are disturbed on a global scale by environmental changes including rising sea surface temperatures and ocean acidification. Little is known about how corals respond or adapt to these environmental changes especially at the molecular level. This is mostly because of the paucity of genome-wide studies on corals and the application of systems approaches that incorporate the latter. Like in any other organism, the response of corals to stress is tightly controlled by the coordinated interplay of many transcription factors. Results Here, we develop and apply a new system-wide approach in order to infer combinatorial transcription factor networks of the reef-building coral Acropora millepora. By integrating sequencing-derived transcriptome measurements, a network of physically interacting transcription factors, and phylogenetic network footprinting we were able to infer such a network. Analysis of the network across a phylogenetically broad sample of five species, including human, reveals that despite the apparent simplicity of corals, their transcription factors repertoire and interaction networks seem to be largely conserved. In addition, we were able to identify interactions among transcription factors that appear to be species-specific lending strength to the novel concept of "Taxonomically Restricted Interactions". Conclusions This study provides the first look at transcription factor networks in corals. We identified a transcription factor repertoire encoded by the coral genome and found consistencies of the domain architectures of transcription factors and conserved regulatory subnetworks across eumetazoan species, providing insight into how regulatory networks have evolved. PMID:21526989

  17. Unexpectedly complex gradation of coral population structure in the Nansei Islands, Japan.

    PubMed

    Zayasu, Yuna; Nakajima, Yuichi; Sakai, Kazuhiko; Suzuki, Go; Satoh, Noriyuki; Shinzato, Chuya

    2016-08-01

    To establish effective locations and sizes of potential protected areas for reef ecosystems, detailed information about source and sink relationships between populations is critical, especially in archipelagic regions. Therefore, we assessed population structure and genetic diversity of Acropora tenuis, one of the dominant stony coral species in the Pacific, using 13 microsatellite markers to investigate 298 colonies from 15 locations across the Nansei Islands in southwestern Japan. Genetic diversity was not significant among sampling locations, even in possibly peripheral locations. In addition, our results showed that there are at least two populations of A. tenuis in the study area. The level of genetic differentiation between these populations was relatively low, but significant between many pairs of sampling locations. Directions of gene flow, which were estimated using a coalescence-based approach, suggest that gene flow not only occurs from south to north, but also from north to south in various locations. Consequently, the Yaeyama Islands and the Amami Islands are potential northern and southern sources of corals. On the other hand, the Miyako Islands and west central Okinawa Island are potential sink populations. The Kerama Islands and the vicinity of Taketomi Island are potential contact points of genetic subdivision of coral populations in the Nansei Islands. We found that genetic population structure of A. tenuis in the Nansei Islands is more complex than previously thought. These cryptic populations are very important for preserving genetic diversity and should be maintained. PMID:27551399

  18. Revealing the hidden language of complex networks.

    PubMed

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

    2014-01-01

    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. PMID:24686408

  19. Revealing the Hidden Language of Complex Networks

    NASA Astrophysics Data System (ADS)

    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.

  20. Revealing the Hidden Language of Complex Networks

    PubMed Central

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

    2014-01-01

    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. PMID:24686408

  1. Modeling and experiment reveal an unexpected stereoelectronic effect on conformation and scalar couplings of alpha-aminoorganostannanes, with possible relevance to the tin-lithium exchange reaction.

    PubMed

    Santiago, Marcelina; Low, Eddy; Chambournier, Gilles; Gawley, Robert E

    2003-10-31

    The solution conformation of N-methyl-2-(tributylstannyl)piperidines has been determined through the use of vicinal 119Sn-13C coupling constants, revealing a conformational distortion caused by an unexpected stereoelectronic effect in some cases. Specifically, the "equatorial" conformer is distorted into a half-chair, in which the nitrogen lone pair eclipses the C-Sn bond. This distortion, which "costs" approximately 1 kcal/mol, correlates with a conformational dependence of geminal 119Sn-15N couplings and a possible correlation with reactivity in the tin-lithium exchange reaction. PMID:14575474

  2. High-resolution geophysics revealing an unexpected post-Pannonian uplift structure: Schützen continued (Northern Burgenland, Austria)

    NASA Astrophysics Data System (ADS)

    Scheibz, Jürgen; Häusler, Hermann; Kardeis, Gerald

    2010-05-01

    The village Schützen am Gebirge is situated between the Leithagebirge and the Rust Range in the northern Burgenland. The pre-Miocene basement of both ridges is partly covered by marine limestone and clastic sediments of Badenian to Sarmatian age, followed up by Pannonian lacustrine silt- and claystone. First geophysical investigations revealed folding structures in this area (Kollmann et al., 1990). The complex tectonic structure was investigated in a northwest trending section by Scheibz (2006) who clearly demonstrated that the Badenian limestone of the Kalkofen quarry north of Schützen is a horst structure within a pronounced antiform. Whereas an extensional regime prevailed during the Pannonian, local post-Pannonian compression was postulated forming the syn- and anticline structures north of Schützen (Häusler et al., 2007; Häusler et al., 2010). In order to study the surroundings of the "Kalkofen-anticline", additional investigations were conducted. Four 2D electrical resistivity tomography (ERT) profiles, each 1000 - 2000 meters long, allowed for subsurface mapping the Kalkofen-structure as a very narrow zone. Furthermore two sites in the center of the anticline structure were investigated by a raster of fifteen high-resolution 2D-ERT sections ("Kalkofen" site and "sports field" site, situated about 400 southwest of the Kalkofen site). Six profiles at the Kalkofen site revealed a northeast trending lens-shaped high-resistivity zone consisting of (Badenian) limestone down to a depth of approximately ten meters, which is underlain by low resistivity beds (of Pannonian age) down to thirty meters. Nine shallow high-resolution profiles at the sports field site show resistivity patterns matching the Leithakalk-limestone down to a depth of only five meters. Additionally a high-resolution 3D ERT block, about 8.600 m2 in size, was measured in the center of the sports field site. Again, high-resistivity beds interpreted as Miocene limestone down to a depth of 25

  3. A neutral branched platinum-acetylide complex possessing a tetraphenylethylene core: preparation of a luminescent organometallic gelator and its unexpected spectroscopic behaviour during sol-to-gel transition.

    PubMed

    Ren, Yuan-Yuan; Wu, Nai-Wei; Huang, Junhai; Xu, Zheng; Sun, Dan-Dan; Wang, Cui-Hong; Xu, Lin

    2015-10-21

    A neutral branched platinum-acetylide complex TPA possessing a tetraphenylethylene core was successfully prepared, which was found to form luminescent organometallic gels in ethyl acetate. Stimulated by temperature or F(-), the reversible gel-sol transition was realized. More interestingly, TPA exhibited an unexpected blue shift of the emission during the sol-to-gel transition. PMID:26323961

  4. Second generation sequencing and morphological faecal analysis reveal unexpected foraging behaviour by Myotis nattereri (Chiroptera, Vespertilionidae) in winter

    PubMed Central

    2014-01-01

    Background Temperate winters produce extreme energetic challenges for small insectivorous mammals. Some bat species inhabiting locations with mild temperate winters forage during brief inter-torpor normothermic periods of activity. However, the winter diet of bats in mild temperate locations is studied infrequently. Although microscopic analyses of faeces have traditionally been used to characterise bat diet, recently the coupling of PCR with second generation sequencing has offered the potential to further advance our understanding of animal dietary composition and foraging behaviour by allowing identification of a much greater proportion of prey items often with increased taxonomic resolution. We used morphological analysis and Illumina-based second generation sequencing to study the winter diet of Natterer’s bat (Myotis nattereri) and compared the results obtained from these two approaches. For the first time, we demonstrate the applicability of the Illumina MiSeq platform as a data generation source for bat dietary analyses. Results Faecal pellets collected from a hibernation site in southern England during two winters (December-March 2009–10 and 2010–11), indicated that M. nattereri forages throughout winter at least in a location with a mild winter climate. Through morphological analysis, arthropod fragments from seven taxonomic orders were identified. A high proportion of these was non-volant (67.9% of faecal pellets) and unexpectedly included many lepidopteran larvae. Molecular analysis identified 43 prey species from six taxonomic orders and confirmed the frequent presence of lepidopteran species that overwinter as larvae. Conclusions The winter diet of M. nattereri is substantially different from other times of the year confirming that this species has a wide and adaptable dietary niche. Comparison of DNA derived from the prey to an extensive reference dataset of potential prey barcode sequences permitted fine scale taxonomic resolution of prey

  5. Culture-independent genome sequencing of clinical samples reveals an unexpected heterogeneity of infections by Chlamydia pecorum.

    PubMed

    Bachmann, Nathan L; Sullivan, Mitchell J; Jelocnik, Martina; Myers, Garry S A; Timms, Peter; Polkinghorne, Adam

    2015-05-01

    Chlamydia pecorum is an important global pathogen of livestock, and it is also a significant threat to the long-term survival of Australia's koala populations. This study employed a culture-independent DNA capture approach to sequence C. pecorum genomes directly from clinical swab samples collected from koalas with chlamydial disease as well as from sheep with arthritis and conjunctivitis. Investigations into single-nucleotide polymorphisms within each of the swab samples revealed that a portion of the reads in each sample belonged to separate C. pecorum strains, suggesting that all of the clinical samples analyzed contained mixed populations of genetically distinct C. pecorum isolates. This observation was independent of the anatomical site sampled and the host species. Using the genomes of strains identified in each of these samples, whole-genome phylogenetic analysis revealed that a clade containing a bovine and a koala isolate is distinct from other clades comprised of livestock or koala C. pecorum strains. Providing additional evidence to support exposure of koalas to Australian livestock strains, two minor strains assembled from the koala swab samples clustered with livestock strains rather than koala strains. Culture-independent probe-based genome capture and sequencing of clinical samples provides the strongest evidence yet to suggest that naturally occurring chlamydial infections are comprised of multiple genetically distinct strains. PMID:25740768

  6. Detailed monitoring of a small but recovering population reveals sublethal effects of disease and unexpected interactions with supplemental feeding.

    PubMed

    Tollington, Simon; Greenwood, Andrew; Jones, Carl G; Hoeck, Paquita; Chowrimootoo, Aurélie; Smith, Donal; Richards, Heather; Tatayah, Vikash; Groombridge, Jim J

    2015-07-01

    Infectious diseases are widely recognized to have substantial impact on wildlife populations. These impacts are sometimes exacerbated in small endangered populations, and therefore, the success of conservation reintroductions to aid the recovery of such species can be seriously threatened by outbreaks of infectious disease. Intensive management strategies associated with conservation reintroductions can further compound these negative effects in such populations. Exploring the sublethal effects of disease outbreaks among natural populations is challenging and requires longitudinal, individual life-history data on patterns of reproductive success and other indicators of individual fitness. Long-term monitoring data concerning detailed reproductive information of the reintroduced Mauritius parakeet (Psittacula echo) population collected before, during and after a disease outbreak was investigated. Deleterious effects of an outbreak of beak and feather disease virus (BFDV) were revealed on hatch success, but these effects were remarkably short-lived and disproportionately associated with breeding pairs which took supplemental food. Individual BFDV infection status was not predicted by any genetic, environmental or conservation management factors and was not associated with any of our measures of immune function, perhaps suggesting immunological impairment. Experimental immunostimulation using the PHA (phytohaemagglutinin assay) challenge technique did, however, provoke a significant cellular immune response. We illustrate the resilience of this bottlenecked and once critically endangered, island-endemic species to an epidemic outbreak of BFDV and highlight the value of systematic monitoring in revealing inconspicuous but nonetheless substantial ecological interactions. Our study demonstrates that the emergence of such an infectious disease in a population ordinarily associated with increased susceptibility does not necessarily lead to deleterious impacts on population

  7. An unexpected Schiff base-type Ni(II) complex: Synthesis, crystal structures, fluorescence, electrochemical property and SOD-like activities

    NASA Astrophysics Data System (ADS)

    Chai, Lan-Qin; Zhang, Hong-Song; Huang, Jiao-Jiao; Zhang, Yu-Li

    2015-02-01

    An unexpected Schiff base-type Ni(II) complex, [Ni(L2)2]ṡCH3OH (HL2 = 1-(2-{[(E)-3, 5-dibromo-2-hydroxybenzylidene]amino}phenyl)ethanone oxime), has been synthesized via complexation of Ni(II) acetate tetrahydrate with HL1 (2-(3,5-dibromo-2-hydroxyphenyl)-4-methyl-1,2-dihydroquinazoline 3-oxide) originally. HL1 and its corresponding Ni(II) complex were characterized by IR, 1H NMR spectra, as well as by elemental analysis, UV-Vis and emission spectroscopy, respectively. Crystal structures of the ligand and complex have been determined by single-crystal X-ray diffraction. Each complex links two other molecules into an infinite 1-D chain via intermolecular hydrogen bonding interactions. Moreover, the electrochemical property of the nickle complex was studied by cyclic voltammetry. In addition, SOD-like activities of HL1 and Ni(II) complex were also investigated.

  8. Multilocus phylogenetic analyses reveal unexpected abundant diversity and significant disjunct distribution pattern of the Hedgehog Mushrooms (Hydnum L.)

    PubMed Central

    Feng, Bang; Wang, Xiang-Hua; Ratkowsky, David; Gates, Genevieve; Lee, Su See; Grebenc, Tine; Yang, Zhu L.

    2016-01-01

    Hydnum is a fungal genus proposed by Linnaeus in the early time of modern taxonomy. It contains several ectomycorrhizal species which are commonly consumed worldwide. However, Hydnum is one of the most understudied fungal genera, especially from a molecular phylogenetic view. In this study, we extensively gathered specimens of Hydnum from Asia, Europe, America and Australasia, and analyzed them by using sequences of four gene fragments (ITS, nrLSU, tef1α and rpb1). Our phylogenetic analyses recognized at least 31 phylogenetic species within Hydnum, 15 of which were reported for the first time. Most Australasian species were recognized as strongly divergent old relics, but recent migration between Australasia and the Northern Hemisphere was also detected. Within the Northern Hemisphere, frequent historical biota exchanges between the Old World and the New World via both the North Atlantic Land Bridge and the Bering Land Bridge could be elucidated. Our study also revealed that most Hydnum species found in subalpine areas of the Hengduan Mountains in southwestern China occur in northeastern/northern China and Europe, indicating that the composition of the mycobiota in the Hengduan Mountains reigion is more complicated than what we have known before. PMID:27151256

  9. Multilocus phylogenetic analyses reveal unexpected abundant diversity and significant disjunct distribution pattern of the Hedgehog Mushrooms (Hydnum L.).

    PubMed

    Feng, Bang; Wang, Xiang-Hua; Ratkowsky, David; Gates, Genevieve; Lee, Su See; Grebenc, Tine; Yang, Zhu L

    2016-01-01

    Hydnum is a fungal genus proposed by Linnaeus in the early time of modern taxonomy. It contains several ectomycorrhizal species which are commonly consumed worldwide. However, Hydnum is one of the most understudied fungal genera, especially from a molecular phylogenetic view. In this study, we extensively gathered specimens of Hydnum from Asia, Europe, America and Australasia, and analyzed them by using sequences of four gene fragments (ITS, nrLSU, tef1α and rpb1). Our phylogenetic analyses recognized at least 31 phylogenetic species within Hydnum, 15 of which were reported for the first time. Most Australasian species were recognized as strongly divergent old relics, but recent migration between Australasia and the Northern Hemisphere was also detected. Within the Northern Hemisphere, frequent historical biota exchanges between the Old World and the New World via both the North Atlantic Land Bridge and the Bering Land Bridge could be elucidated. Our study also revealed that most Hydnum species found in subalpine areas of the Hengduan Mountains in southwestern China occur in northeastern/northern China and Europe, indicating that the composition of the mycobiota in the Hengduan Mountains reigion is more complicated than what we have known before. PMID:27151256

  10. Unexpected allelic heterogeneity and spectrum of mutations in Fowler syndrome revealed by next-generation exome sequencing.

    PubMed

    Lalonde, Emilie; Albrecht, Steffen; Ha, Kevin C H; Jacob, Karine; Bolduc, Nathalie; Polychronakos, Constantin; Dechelotte, Pierre; Majewski, Jacek; Jabado, Nada

    2010-08-01

    Protein coding genes constitute approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease-related traits. Therefore, efficient strategies for selectively sequencing complete coding regions (i.e., "whole exome") have the potential to contribute our understanding of human diseases. We used a method for whole-exome sequencing coupling Agilent whole-exome capture to the Illumina DNA-sequencing platform, and investigated two unrelated fetuses from nonconsanguineous families with Fowler Syndrome (FS), a stereotyped phenotype lethal disease. We report novel germline mutations in feline leukemia virus subgroup C cellular-receptor-family member 2, FLVCR2, which has recently been shown to cause FS. Using this technology, we identified three types of genetic abnormalities: point-mutations, insertions-deletions, and intronic splice-site changes (first pathogenic report using this technology), in the fetuses who both were compound heterozygotes for the disease. Although revealing a high level of allelic heterogeneity and mutational spectrum in FS, this study further illustrates the successful application of whole-exome sequencing to uncover genetic defects in rare Mendelian disorders. Of importance, we show that we can identify genes underlying rare, monogenic and recessive diseases using a limited number of patients (n=2), in the absence of shared genetic heritage and in the presence of allelic heterogeneity. PMID:20518025

  11. Unexpected high 35S concentration revealing strong downward transport of stratospheric air during the monsoon transitional period in East Asia

    NASA Astrophysics Data System (ADS)

    Lin, Mang; Zhang, Zhisheng; Su, Lin; Su, Binbin; Liu, Lanzhong; Tao, Jun; Fung, Jimmy C. H.; Thiemens, Mark H.

    2016-03-01

    October is the monsoon transitional period in East Asia (EA) involving a series of synoptic activities that may enhance the downward transport of stratospheric air to the planetary boundary layer (PBL). Here we use cosmogenic 35S in sulfate aerosols (35SO42-) as a tracer for air masses originating from the stratosphere and transported downward to quantify these mixing processes. From 1 year 35SO42- measurements (March 2014 to February 2015) at a background station in EA we find remarkably enhanced 35SO42- concentration (3150 atoms m-3) in October, the highest value ever reported for natural sulfate aerosols. A four-box 1-D model and meteorological analysis reveal that strong downward transport from the free troposphere is a vital process entraining aged stratospheric air masses to the PBL. The aged stratospheric masses are accumulated in the PBL, accelerating the SO2 transformation to SO42-. Implications for the tropospheric O3 budget and the CO2 biogeochemical cycle are discussed.

  12. Multilocus sequence analysis of Brazilian Rhizobium microsymbionts of common bean (Phaseolus vulgaris L.) reveals unexpected taxonomic diversity.

    PubMed

    Ribeiro, Renan Augusto; Barcellos, Fernando Gomes; Thompson, Fabiano L; Hungria, Mariangela

    2009-05-01

    The diazotrophic bacteria collectively known as "rhizobia" are important for establishing symbiotic N(2)-fixing associations with many legumes. These microbes have been used for over a century as an environmentally beneficial and cost-effective means of ensuring acceptable yields of agricultural legumes. The most widely used phylogenetic marker for identification and classification of rhizobia has been the 16S rRNA gene; however, this marker fails to discriminate some closely related species. In this study, we established the first multilocus sequence analysis (MLSA) scheme for the identification and classification of rhizobial microsymbionts of common bean (Phaseolus vulgaris L.). We analyzed 12 Brazilian strains representative of a collection of over 850 isolates in addition to type and reference rhizobial strains, by sequencing recA, dnaK, gltA, glnII and rpoA genes. Gene sequence similarities among the five type/reference Rhizobium strains which are symbionts of common bean ranged from 95 to 100% for 16S rRNA, and from 83 to 99% for the other five genes. Rhizobial species described as symbionts of common bean also formed separate groups upon analysis of single and concatenated gene sequences, and clusters formed in each tree were in good mutual agreement. The five additional loci may thus be considered useful markers of the genus Rhizobium; in addition, MLSA also revealed broad genetic diversity among strains classified as Rhizobium tropici, providing evidence of new species. PMID:19403105

  13. Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles.

    PubMed

    Petrényi, Katalin; Molero, Cristina; Kónya, Zoltán; Erdődi, Ferenc; Ariño, Joaquin; Dombrádi, Viktor

    2016-01-01

    Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a

  14. Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles

    PubMed Central

    Petrényi, Katalin; Molero, Cristina; Kónya, Zoltán; Erdődi, Ferenc; Ariño, Joaquin; Dombrádi, Viktor

    2016-01-01

    Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a

  15. Lineage analysis of basal epithelial cells reveals their unexpected plasticity and supports a cell-of-origin model for prostate cancer heterogeneity.

    PubMed

    Wang, Zhu A; Mitrofanova, Antonina; Bergren, Sarah K; Abate-Shen, Cory; Cardiff, Robert D; Califano, Andrea; Shen, Michael M

    2013-03-01

    A key issue in cancer biology is whether oncogenic transformation of different cell types of origin within an adult tissue gives rise to distinct tumour subtypes that differ in their prognosis and/or treatment response. We now show that initiation of prostate tumours in basal or luminal epithelial cells in mouse models results in tumours with distinct molecular signatures that are predictive of human patient outcomes. Furthermore, our analysis of untransformed basal cells reveals an unexpected assay dependence of their stem cell properties in sphere formation and transplantation assays versus genetic lineage tracing during prostate regeneration and adult tissue homeostasis. Although oncogenic transformation of basal cells gives rise to tumours with luminal phenotypes, cross-species bioinformatic analyses indicate that tumours of luminal origin are more aggressive than tumours of basal origin, and identify a molecular signature associated with patient outcome. Our results reveal the inherent plasticity of basal cells, and support a model in which different cells of origin generate distinct molecular subtypes of prostate cancer. PMID:23434823

  16. Unexpected Actinyl Cation-Directed Structural Variation in Neptunyl(VI) A-Type Tri-lacunary Heteropolyoxotungstate Complexes.

    PubMed

    Berg, John M; Gaunt, Andrew J; May, Iain; Pugmire, Alison L; Reilly, Sean D; Scott, Brian L; Wilkerson, Marianne P

    2015-05-01

    A-type tri-lacunary heteropolyoxotungstate anions (e.g., [PW9O34](9-), [AsW9O34](9-), [SiW9O34](10-), and [GeW9O34](10-)) are multidentate oxygen donor ligands that readily form sandwich complexes with actinyl cations ({UO2}(2+), {NpO2}(+), {NpO2}(2+), and {PuO2}(2+)) in near-neutral/slightly alkaline aqueous solutions. Two or three actinyl cations are sandwiched between two tri-lacunary anions, with additional cations (Na(+), K(+), or NH4(+)) also often held within the cluster. Studies thus far have indicated that it is these additional +1 cations, rather than the specific actinyl cation, that direct the structural variation in the complexes formed. We now report the structural characterization of the neptunyl(VI) cluster complex (NH4)13[Na(NpO2)2(A-α-PW9O34)2]·12H2O. The anion in this complex, [Na(NpO2)2(PW9O34)2](13-), contains one Na(+) cation and two {NpO2}(2+) cations held between two [PW9O34](9-) anions, with an additional partial occupancy NH4(+) or {NpO2}(2+) cation also present. In the analogous uranium(VI) system, under similar reaction conditions that include an excess of NH4Cl in the parent solution, it was previously shown that [(NH4)2(U(VI)O2)2(A-PW9O34)2](12-) is the dominant species in both solution and the crystallized salt. Spectroscopic studies provide further proof of differences in the observed chemistry for the {NpO2}(2+)/[PW9O34](9-) and {UO2}(2+)/[PW9O34](9-) systems, both in solution and in solid state complexes crystallized from comparable salt solutions. This work reveals that varying the actinide element (Np vs U) can indeed measurably impact structure and complex stability in the cluster chemistry of actinyl(VI) cations with A-type tri-lacunary heteropolyoxotungstate anions. PMID:25901900

  17. Unexpected Actinyl Cation-Directed Structural Variation in Neptunyl(VI) A-Type Tri-lacunary Heteropolyoxotungstate Complexes

    DOE PAGESBeta

    Berg, John M.; Gaunt, Andrew J.; May, Iain; Pugmire, Alison L.; Reilly, Sean D.; Scott, Brian L.; Wilkerson, Marianne P.

    2015-04-22

    A-type tri-lacunary heteropolyoxotungstate anions (e.g., [PW9O34]9-, [AsW9O34]9-, [SiW9O34]10- and [GeW9O34]10-) are multi-dentate oxygen donor ligands that readily form sandwich complexes with actinyl cations ({UO2}2+, {NpO2}+, {NpO2}2+ & {PuO2}2+) in near neutral/slightly alkaline aqueous solutions. Two or three actinyl cations are sandwiched between two trilacunary anions, with additional cations (Na+, K+ or NH4 +) also often held within the cluster. Studies thus far have indicated that it is these additional +I cations, rather than the specific actinyl cation, that direct the structural variation in the complexes formed. We now report the structural characterization of the neptunyl (VI) cluster complex (NH4)13 [Na(NpO2)2(A-α-more » PW9O34)2]·12H2O. The anion in this complex, [Na(NpO2)2(PW9O34)2]13-, contains one Na+ cation and two {NpO2}2+ cations held between two [PW9O34]9- anions – with an additional partial occupancy NH4 + or {NpO2}2+ cation also present. In the analogous uranium (VI) system, under similar reaction conditions that includes an excess of NH4Cl in the parent solution, it was previously shown that [(NH4)2(UVIO2)2(A-PW9O34)2]12- is the dominant species in both solution and the crystallized salt. Spectroscopic studies provide further proof of differences in the observed chemistry for the {NpO2}2+/[PW9O34]9- and {UO2}2+/[PW9O34]9- systems, both in solution and in solid state complexes crystallized from comparable salt solutions. The work revealed that varying the actinide element (Np vs. U) can indeed measurably impact structure and complex stability in the cluster chemistry of actinyl (VI) cations with A-type tri-lacunary heteropolyoxotungstate anions.« less

  18. Adaptation to High Ethanol Reveals Complex Evolutionary Pathways.

    PubMed

    Voordeckers, Karin; Kominek, Jacek; Das, Anupam; Espinosa-Cantú, Adriana; De Maeyer, Dries; Arslan, Ahmed; Van Pee, Michiel; van der Zande, Elisa; Meert, Wim; Yang, Yudi; Zhu, Bo; Marchal, Kathleen; DeLuna, Alexander; Van Noort, Vera; Jelier, Rob; Verstrepen, Kevin J

    2015-11-01

    Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts. PMID:26545090

  19. Adaptation to High Ethanol Reveals Complex Evolutionary Pathways

    PubMed Central

    Das, Anupam; Espinosa-Cantú, Adriana; De Maeyer, Dries; Arslan, Ahmed; Van Pee, Michiel; van der Zande, Elisa; Meert, Wim; Yang, Yudi; Zhu, Bo; Marchal, Kathleen; DeLuna, Alexander; Van Noort, Vera; Jelier, Rob; Verstrepen, Kevin J.

    2015-01-01

    Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts. PMID:26545090

  20. Unexpected Actinyl Cation-Directed Structural Variation in Neptunyl(VI) A-Type Tri-lacunary Heteropolyoxotungstate Complexes

    SciTech Connect

    Berg, John M.; Gaunt, Andrew J.; May, Iain; Pugmire, Alison L.; Reilly, Sean D.; Scott, Brian L.; Wilkerson, Marianne P.

    2015-04-22

    >/[PW9O34]9- and {UO2}2+/[PW9O34]9- systems, both in solution and in solid state complexes crystallized from comparable salt solutions. The work revealed that varying the actinide element (Np vs. U) can indeed measurably impact structure and complex stability in the cluster chemistry of actinyl (VI) cations with A-type tri-lacunary heteropolyoxotungstate anions.

  1. Genome-Wide Analysis Reveals a Major Role in Cell Fate Maintenance and an Unexpected Role in Endoreduplication for the Drosophila FoxA Gene Fork Head

    PubMed Central

    Maruyama, Rika; Grevengoed, Elizabeth; Stempniewicz, Peter; Andrew, Deborah J.

    2011-01-01

    Transcription factors drive organogenesis, from the initiation of cell fate decisions to the maintenance and implementation of these decisions. The Drosophila embryonic salivary gland provides an excellent platform for unraveling the underlying transcriptional networks of organ development because Drosophila is relatively unencumbered by significant genetic redundancy. The highly conserved FoxA family transcription factors are essential for various aspects of organogenesis in all animals that have been studied. Here, we explore the role of the single Drosophila FoxA protein Fork head (Fkh) in salivary gland organogenesis using two genome-wide strategies. A large-scale in situ hybridization analysis reveals a major role for Fkh in maintaining the salivary gland fate decision and controlling salivary gland physiological activity, in addition to its previously known roles in morphogenesis and survival. The majority of salivary gland genes (59%) are affected by fkh loss, mainly at later stages of salivary gland development. We show that global expression of Fkh cannot drive ectopic salivary gland formation. Thus, unlike the worm FoxA protein PHA-4, Fkh does not function to specify cell fate. In addition, Fkh only indirectly regulates many salivary gland genes, which is also distinct from the role of PHA-4 in organogenesis. Our microarray analyses reveal unexpected roles for Fkh in blocking terminal differentiation and in endoreduplication in the salivary gland and in other Fkh-expressing embryonic tissues. Overall, this study demonstrates an important role for Fkh in determining how an organ preserves its identity throughout development and provides an alternative paradigm for how FoxA proteins function in organogenesis. PMID:21698206

  2. Surprise maximization reveals the community structure of complex networks

    NASA Astrophysics Data System (ADS)

    Aldecoa, Rodrigo; Marín, Ignacio

    2013-01-01

    How to determine the community structure of complex networks is an open question. It is critical to establish the best strategies for community detection in networks of unknown structure. Here, using standard synthetic benchmarks, we show that none of the algorithms hitherto developed for community structure characterization perform optimally. Significantly, evaluating the results according to their modularity, the most popular measure of the quality of a partition, systematically provides mistaken solutions. However, a novel quality function, called Surprise, can be used to elucidate which is the optimal division into communities. Consequently, we show that the best strategy to find the community structure of all the networks examined involves choosing among the solutions provided by multiple algorithms the one with the highest Surprise value. We conclude that Surprise maximization precisely reveals the community structure of complex networks.

  3. Surprise maximization reveals the community structure of complex networks.

    PubMed

    Aldecoa, Rodrigo; Marín, Ignacio

    2013-01-01

    How to determine the community structure of complex networks is an open question. It is critical to establish the best strategies for community detection in networks of unknown structure. Here, using standard synthetic benchmarks, we show that none of the algorithms hitherto developed for community structure characterization perform optimally. Significantly, evaluating the results according to their modularity, the most popular measure of the quality of a partition, systematically provides mistaken solutions. However, a novel quality function, called Surprise, can be used to elucidate which is the optimal division into communities. Consequently, we show that the best strategy to find the community structure of all the networks examined involves choosing among the solutions provided by multiple algorithms the one with the highest Surprise value. We conclude that Surprise maximization precisely reveals the community structure of complex networks. PMID:23320141

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

    SciTech Connect

    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.

  5. Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate–peptide complex structures

    PubMed Central

    Zoll, Sebastian; Stanchev, Stancho; Began, Jakub; Škerle, Jan; Lepšík, Martin; Peclinovská, Lucie; Majer, Pavel; Strisovsky, Kvido

    2014-01-01

    The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the ‘water retention site’, suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG. PMID:25216680

  6. Fundamental Differences between Group 8 Metals: Unexpected Oxidation State Preferences and Mechanisms in Ruthenium Borylene Complex Formation.

    PubMed

    Braunschweig, Holger; Damme, Alexander; Dewhurst, Rian D; Radacki, Krzysztof; Weißenberger, Felix; Wennemann, Benedikt; Ye, Qing

    2016-06-13

    The reaction of the salts K[Ru(CO)3 (PMe3 )(SiR3 )] (R=Me, Et) with Br2 BDur or Cl2 BDur (Dur=2,3,5,6-Me4 C6 H) leads to both boryl and borylene complexes of divalent ruthenium, the former through simple salt elimination and the latter through subsequent CO loss and 1,2-halide shift. The balance of products can be altered by varying the reaction conditions; boryl complexes can be favored by the addition of CO, and borylene complexes by removal of CO under vacuum. All of these products are in competition with the corresponding (aryl)(halo)(trialkylsilyl)borane, a reductive elimination product. The Ru(II) borylene products and the mechanisms that form them are distinctly different from the analogous reactions with iron, which lead to low-valent borylene complexes, highlighting fundamental differences in oxidation state preferences between iron and ruthenium. PMID:27124888

  7. Investigation of LKB1 Ser431 phosphorylation and Cys433 farnesylation using mouse knockin analysis reveals an unexpected role of prenylation in regulating AMPK activity

    PubMed Central

    Houde, Vanessa P.; Ritorto, Maria Stella; Gourlay, Robert; Varghese, Joby; Davies, Paul; Shpiro, Natalia; Sakamoto, Kei; Alessi, Dario R.

    2013-01-01

    The LKB1 tumour suppressor protein kinase functions to activate two isoforms of AMPK (AMP-activated protein kinase) and 12 members of the AMPK-related family of protein kinases. The highly conserved C-terminal residues of LKB1 are phosphorylated (Ser431) by PKA (cAMP-dependent protein kinase) and RSK (ribosomal S6 kinase) and farnesylated (Cys433) within a CAAX motif. To better define the role that these post-translational modifications play, we created homozygous LKB1S431A/S431A and LKB1C433S/C433S knockin mice. These animals were viable, fertile and displayed no overt phenotypes. Employing a farnesylation-specific monoclonal antibody that we generated, we established by immunoprecipitation that the vast majority, if not all, of the endogenous LKB1 is prenylated. Levels of LKB1 localized at the membrane of the liver of LKB1C433S/C433S mice and their fibroblasts were reduced substantially compared with the wild-type mice, confirming that farnesylation plays a role in mediating membrane association. Although AMPK was activated normally in the LKB1S431A/S431A animals, we unexpectedly observed in all of the examined tissues and cells taken from LKB1C433S/C433S mice that the basal, as well as that induced by the AMP-mimetic AICAR (5-amino-4-imidazolecarboxamide riboside), AMPK activation, phenformin and muscle contraction were significantly blunted. This resulted in a reduced ability of AICAR to inhibit lipid synthesis in primary hepatocytes isolated from LKB1C433S/C433S mice. The activity of several of the AMPK-related kinases analysed [BRSK1 (BR serine/threonine kinase 1), BRSK2, NUAK1 (NUAK family, SNF1-like kinase 1), SIK3 (salt-inducible kinase 3) and MARK4 (MAP/microtubule affinity-regulating kinase 4)] was not affected in tissues derived from LKB1S431A/S431A or LKB1C433S/C433S mice. Our observations reveal for the first time that farnesylation of LKB1 is required for the activation of AMPK. Previous reports have indicated that a pool of AMPK is localized at the

  8. Unexpected Efficiency of a Luminescent Samarium(III) Complex for Combined Visible and Near-Infrared Biphotonic Microscopy.

    PubMed

    Bui, Anh Thy; Grichine, Alexei; Brasselet, Sophie; Duperray, Alain; Andraud, Chantal; Maury, Olivier

    2015-12-01

    An original samarium(III) complex based on a triazacyclononane platform functionalized with a charge-transfer antenna chromophore exhibited optimized brightness and was successfully used as an emissive species for two-photon microscopy experiments in both the visible and near-infrared spectral ranges. PMID:26489885

  9. Graph analysis of cortical networks reveals complex anatomical communication substrate

    NASA Astrophysics Data System (ADS)

    Zamora-López, Gorka; Zhou, Changsong; Kurths, Jürgen

    2009-03-01

    Sensory information entering the nervous system follows independent paths of processing such that specific features are individually detected. However, sensory perception, awareness, and cognition emerge from the combination of information. Here we have analyzed the corticocortical network of the cat, looking for the anatomical substrate which permits the simultaneous segregation and integration of information in the brain. We find that cortical communications are mainly governed by three topological factors of the underlying network: (i) a large density of connections, (ii) segregation of cortical areas into clusters, and (iii) the presence of highly connected hubs aiding the multisensory processing and integration. Statistical analysis of the shortest paths reveals that, while information is highly accessible to all cortical areas, the complexity of cortical information processing may arise from the rich and intricate alternative paths in which areas can influence each other.

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

    EPA Science Inventory

    We used an extensive dataset of remotely sensed summertime river temperature to compare longitudinal profiles (temperature versus distance) for 54 rivers in the Pacific Northwest. We evaluated (1) how often profiles fit theoretical expectations of asymptotic downstream warming, a...

  11. A complex North Atlantic permanent pycnocline revealed by Argo data

    NASA Astrophysics Data System (ADS)

    Feucher, Charlène; Maze, Guillaume; Mercier, Herlé

    2015-04-01

    In the North Atlantic subtropical gyre, the oceanic vertical structure of density is characterized by a region of rapid increase with depth. This layer is called the permanent pycnocline. The pycnocline is the transition layer between light, low-latitude, surface water masses which are ventilated every winter when penetrated locally by the mixed layer and dense, deeper water masses whose properties are set in the high latitudes. Assessing the structure and variability of the permanent pycnocline is of a major interest in the understanding of the climate system because the pycnocline embeds the warm water sphere and most of the wind-forced horizontal circulation. We characterized the large scale structure of the permanent pycnocline with in-situ data from the Argo array. We developed a new method to objectively characterize its properties (depth, thickness, temperature, salinity, density, potential vorticity). Results reveal a surprisingly complex structure with inhomogeneous properties. In the Gulf Stream recirculation region the pycnocline is deep, thick, the maximum of stratification is found in the middle on the layer and follow an isopycnal surface. But away from this textbook regional description, the pycnocline is characterized by vertical asymmetries and gradients in thermohaline properties. T/S distribution along the permanent pycnocline depth reveals a diversity of water masses. We will present the mean observed structure and properties of the permanent pycnocline and relate them to physical processes that constraint them.

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

    PubMed

    Alshamsi, Aamena; Pianesi, Fabio; Lepri, Bruno; Pentland, Alex; Rahwan, Iyad

    2015-01-01

    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. PMID:26313449

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

    PubMed Central

    Alshamsi, Aamena; Pianesi, Fabio; Lepri, Bruno; Pentland, Alex; Rahwan, Iyad

    2015-01-01

    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. PMID:26313449

  14. Thermodynamic and Kinetic Characterization of the Protein Z-dependent Protease Inhibitor (ZPI)-Protein Z Interaction Reveals an Unexpected Role for ZPI Lys-239*

    PubMed Central

    Huang, Xin; Zhou, Jian; Zhou, Aiwu; Olson, Steven T.

    2015-01-01

    The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), circulates in blood as a tight complex with its cofactor, protein Z (PZ), enabling it to function as a rapid inhibitor of membrane-associated factor Xa. Here, we show that N,N′-dimethyl-N-(acetyl)-N′-(7-nitrobenz-3-oxa-1,3-diazol-4-yl)ethylenediamine (NBD)-fluorophore-labeled K239C ZPI is a sensitive, moderately perturbing reporter of the ZPI-PZ interaction and utilize the labeled ZPI to characterize in-depth the thermodynamics and kinetics of wild-type and variant ZPI-PZ interactions. NBD-labeled K239C ZPI bound PZ with ∼3 nm KD and ∼400% fluorescence enhancement at physiologic pH and ionic strength. The NBD-ZPI-PZ interaction was markedly sensitive to ionic strength and pH but minimally affected by temperature, consistent with the importance of charged interactions. NBD-ZPI-PZ affinity was reduced ∼5-fold by physiologic calcium levels to resemble NBD-ZPI affinity for γ-carboxyglutamic acid/EGF1-domainless PZ. Competitive binding studies with ZPI variants revealed that in addition to previously identified Asp-293 and Tyr-240 hot spot residues, Met-71, Asp-74, and Asp-238 made significant contributions to PZ binding, whereas Lys-239 antagonized binding. Rapid kinetic studies indicated a multistep binding mechanism with diffusion-limited association and slow complex dissociation. ZPI complexation with factor Xa or cleavage decreased ZPI-PZ affinity 2–7-fold by increasing the rate of PZ dissociation. A catalytic role for PZ was supported by the correlation between a decreased rate of PZ dissociation from the K239A ZPI-PZ complex and an impaired ability of PZ to catalyze the K239A ZPI-factor Xa reaction. Together, these results reveal the energetic basis of the ZPI-PZ interaction and suggest an important role for ZPI Lys-239 in PZ catalytic action. PMID:25713144

  15. The unexpected case of reactions of halogens and interhalogens with halide substituted Pd(ii) σ-butadienyl complexes.

    PubMed

    Scattolin, Thomas; Visentin, Fabiano; Santo, Claudio; Bertolasi, Valerio; Canovese, Luciano

    2016-07-28

    We have experimentally studied and theoretically interpreted the addition under stoichiometric conditions of halogens or interhalogens to σ-butadienyl palladium complexes bearing the heteroditopic thioquinolines as spectator ligands. The observed reactions do not involve the expected extrusion of the butadienyl fragment but rather the unpredictable substitution of the halide coordinated to palladium and in some cases also of that bound to the terminal butadienyl carbon. We have explained this peculiar reactivity with a mechanistic hypothesis based on a sequence of selective processes of oxidative addition and reductive elimination involving Pd(iv) intermediates. PMID:27357221

  16. The Dictyostelium prestalk inducer differentiation-inducing factor-1 (DIF-1) triggers unexpectedly complex global phosphorylation changes

    PubMed Central

    Sugden, Chris; Urbaniak, Michael D.; Araki, Tsuyoshi; Williams, Jeffrey G.

    2015-01-01

    Differentiation-inducing factor-1 (DIF-1) is a polyketide that induces Dictyostelium amoebae to differentiate as prestalk cells. We performed a global quantitative screen for phosphorylation changes that occur within the first minutes after addition of DIF-1, using a triple-label SILAC approach. This revealed a new world of DIF-1–controlled signaling, with changes in components of the MAPK and protein kinase B signaling pathways, components of the actinomyosin cytoskeletal signaling networks, and a broad range of small GTPases and their regulators. The results also provide evidence that the Ca2+/calmodulin–dependent phosphatase calcineurin plays a role in DIF-1 signaling to the DimB prestalk transcription factor. At the global level, DIF-1 causes a major shift in the phosphorylation/dephosphorylation equilibrium toward net dephosphorylation. Of interest, many of the sites that are dephosphorylated in response to DIF-1 are phosphorylated in response to extracellular cAMP signaling. This accords with studies that suggest an antagonism between the two inducers and also with the rapid dephosphorylation of the cAMP receptor that we observe in response to DIF-1 and with the known inhibitory effect of DIF-1 on chemotaxis to cAMP. All MS data are available via ProteomeXchange with identifier PXD001555. PMID:25518940

  17. Delving into the complexity of hereditary spastic paraplegias: how unexpected phenotypes and inheritance modes are revolutionizing their nosology.

    PubMed

    Tesson, Christelle; Koht, Jeanette; Stevanin, Giovanni

    2015-06-01

    Hereditary spastic paraplegias (HSP) are rare neurodegenerative diseases sharing the degeneration of the corticospinal tracts as the main pathological characteristic. They are considered one of the most heterogeneous neurological disorders. All modes of inheritance have been described for the 84 different loci and 67 known causative genes implicated up to now. Recent advances in molecular genetics have revealed clinico-genetic heterogeneity of these disorders including their clinical and genetic overlap with other diseases of the nervous system. The systematic analysis of a large set of genes, including exome sequencing, is unmasking unusual phenotypes or inheritance modes associated with mutations in HSP genes and related genes involved in various neurological diseases. A new nosology may emerge after integration and understanding of these new data to replace the current classification. Collectively, functions of the known genes implicate the disturbance of intracellular membrane dynamics and trafficking as the consequence of alterations of cytoskeletal dynamics, lipid metabolism and organelle structures, which represent in fact a relatively small number of cellular processes that could help to find common curative approaches, which are still lacking. PMID:25758904

  18. Geometric Mechanics Reveals Optimal Complex Terrestrial Undulation Patterns

    NASA Astrophysics Data System (ADS)

    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.

  19. An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

    SciTech Connect

    Cook, William J; Senkovich, Olga; Chattopadhyay, Debasish

    2009-06-08

    The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2{angstrom} resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation

  20. Global ‘bootprinting’ reveals the elastic architecture of the yeast TFIIIB–TFIIIC transcription complex in vivo

    PubMed Central

    Nagarajavel, V.; Iben, James R.; Howard, Bruce H.; Maraia, Richard J.; Clark, David J.

    2013-01-01

    TFIIIB and TFIIIC are multi-subunit factors required for transcription by RNA polymerase III. We present a genome-wide high-resolution footprint map of TFIIIB–TFIIIC complexes in Saccharomyces cerevisiae, obtained by paired-end sequencing of micrococcal nuclease-resistant DNA. On tRNA genes, TFIIIB and TFIIIC form stable complexes with the same distinctive occupancy pattern but in mirror image, termed ‘bootprints’. Global analysis reveals that the TFIIIB–TFIIIC transcription complex exhibits remarkable structural elasticity: tRNA genes vary significantly in length but remain protected by TFIIIC. Introns, when present, are markedly less protected. The RNA polymerase III transcription terminator is flexibly accommodated within the transcription complex and, unexpectedly, plays a major structural role by delimiting its 3′-boundary. The ETC sites, where TFIIIC binds without TFIIIB, exhibit different bootprints, suggesting that TFIIIC forms complexes involving other factors. We confirm six ETC sites and report a new site (ETC10). Surprisingly, TFIIIC, but not TFIIIB, interacts with some centromeric nucleosomes, suggesting that interactions between TFIIIC and the centromere may be important in the 3D organization of the nucleus. PMID:23856458

  1. Natural Product Screening Reveals Naphthoquinone Complex I Bypass Factors.

    PubMed

    Vafai, Scott B; Mevers, Emily; Higgins, Kathleen W; Fomina, Yevgenia; Zhang, Jianming; Mandinova, Anna; Newman, David; Shaw, Stanley Y; Clardy, Jon; Mootha, Vamsi K

    2016-01-01

    Deficiency of mitochondrial complex I is encountered in both rare and common diseases, but we have limited therapeutic options to treat this lesion to the oxidative phosphorylation system (OXPHOS). Idebenone and menadione are redox-active molecules capable of rescuing OXPHOS activity by engaging complex I-independent pathways of entry, often referred to as "complex I bypass." In the present study, we created a cellular model of complex I deficiency by using CRISPR genome editing to knock out Ndufa9 in mouse myoblasts, and utilized this cell line to develop a high-throughput screening platform for novel complex I bypass factors. We screened a library of ~40,000 natural product extracts and performed bioassay-guided fractionation on a subset of the top scoring hits. We isolated four plant-derived 1,4-naphthoquinone complex I bypass factors with structural similarity to menadione: chimaphilin and 3-chloro-chimaphilin from Chimaphila umbellata and dehydro-α-lapachone and dehydroiso-α-lapachone from Stereospermum euphoroides. We also tested a small number of structurally related naphthoquinones from commercial sources and identified two additional compounds with complex I bypass activity: 2-methoxy-1,4-naphthoquinone and 2-methoxy-3-methyl-1,4,-naphthoquinone. The six novel complex I bypass factors reported here expand this class of molecules and will be useful as tool compounds for investigating complex I disease biology. PMID:27622560

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

    PubMed

    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. PMID:26659058

  3. The solution structure of the MANEC-type domain from hepatocyte growth factor activator inhibitor-1 reveals an unexpected PAN/apple domain-type fold.

    PubMed

    Hong, Zebin; Nowakowski, Michal; Spronk, Chris; Petersen, Steen V; Andreasen, Peter A; Koźmiński, Wiktor; Mulder, Frans A A; Jensen, Jan K

    2015-03-01

    A decade ago, motif at N-terminus with eight-cysteines (MANEC) was defined as a new protein domain family. This domain is found exclusively at the N-terminus of >400 multi-domain type-1 transmembrane proteins from animals. Despite the large number of MANEC-containing proteins, only one has been characterized at the protein level: hepatocyte growth factor activator inhibitor-1 (HAI-1). HAI-1 is an essential protein, as knockout mice die in utero due to placental defects. HAI-1 is an inhibitor of matriptase, hepsin and hepatocyte growth factor (HGF) activator, all serine proteases with important roles in epithelial development, cell growth and homoeostasis. Dysregulation of these proteases has been causatively implicated in pathological conditions such as skin diseases and cancer. Detailed functional understanding of HAI-1 and other MANEC-containing proteins is hampered by the lack of structural information on MANEC. Although many MANEC sequences exist, sequence-based database searches fail to predict structural homology. In the present paper, we present the NMR solution structure of the MANEC domain from HAI-1, the first three-dimensional (3D) structure from the MANEC domain family. Unexpectedly, MANEC is a new subclass of the PAN/apple domain family, with its own unifying features, such as two additional disulfide bonds, two extended loop regions and additional α-helical elements. As shown for other PAN/apple domain-containing proteins, we propose a similar active role of the MANEC domain in intramolecular and intermolecular interactions. The structure provides a tool for the further elucidation of HAI-1 function as well as a reference for the study of other MANEC-containing proteins. PMID:25510835

  4. Aminoacyl-tRNA synthetase complexes: molecular multitasking revealed

    PubMed Central

    Hausmann, Corinne D.; Ibba, Michael

    2008-01-01

    The accurate synthesis of proteins, dictated by the corresponding nucleotide sequence encoded in mRNA, is essential for cell growth and survival. Central to this process are the aminoacyl-tRNA synthetases (aaRSs), which provide amino acid substrates for the growing polypeptide chain in the form of aminoacyl-tRNAs. The aaRSs are essential for coupling the correct amino acid and tRNA molecules, but are also known to associate in higher order complexes with proteins involved in processes beyond translation. Multiprotein complexes containing aaRSs are found in all three domains of life playing roles in splicing, apoptosis, viral assembly, and regulation of transcription and translation. An overview of the complexes aaRSs form in all domains of life is presented, demonstrating the extensive network of connections between the translational machinery and cellular components involved in a myriad of essential processes beyond protein synthesis. PMID:18522650

  5. Revealing the Complexity of Community-Campus Interactions

    ERIC Educational Resources Information Center

    Nichols, Naomi Elizabeth; Phipps, David; Gaetz, Stephen; Fisher, Alison L.; Tanguay, Nancy

    2014-01-01

    In this paper, four qualitative case studies capture the complex interplay between the social and structural relations that shape community - academic partnerships. Collaborations begin as relationships among people. They are sustained by institutional structures that recognize and support these relationships. Productive collaborations centralize…

  6. Quantitative Genome-Wide Genetic Interaction Screens Reveal Global Epistatic Relationships of Protein Complexes in Escherichia coli

    PubMed Central

    Kumar, Ashwani; Stewart, Geordie; Samanfar, Bahram; Aoki, Hiroyuki; Wagih, Omar; Vlasblom, James; Phanse, Sadhna; Lad, Krunal; Yeou Hsiung Yu, Angela; Graham, Christopher; Jin, Ke; Brown, Eric; Golshani, Ashkan; Kim, Philip; Moreno-Hagelsieb, Gabriel; Greenblatt, Jack; Houry, Walid A.; Parkinson, John; Emili, Andrew

    2014-01-01

    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. PMID:24586182

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

    PubMed

    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. PMID:24905753

  8. Hierarchicality of Trade Flow Networks Reveals Complexity of Products

    PubMed Central

    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 trillion dollars today. Interestingly, around 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 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. PMID:24905753

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

    PubMed Central

    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. PMID:23942320

  10. Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern

    PubMed Central

    Bouton, Clément; Geldreich, Angèle; Ramel, Laëtitia; Ryabova, Lyubov A.; Dimitrova, Maria; Keller, Mario

    2015-01-01

    The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA. PMID:26162084

  11. From Amazonia to the Atlantic forest: molecular phylogeny of Phyzelaphryninae frogs reveals unexpected diversity and a striking biogeographic pattern emphasizing conservation challenges.

    PubMed

    Fouquet, Antoine; Loebmann, Daniel; Castroviejo-Fisher, Santiago; Padial, José M; Orrico, Victor G D; Lyra, Mariana L; Roberto, Igor Joventino; Kok, Philippe J R; Haddad, Célio F B; Rodrigues, Miguel T

    2012-11-01

    Documenting the Neotropical amphibian diversity has become a major challenge facing the threat of global climate change and the pace of environmental alteration. Recent molecular phylogenetic studies have revealed that the actual number of species in South American tropical forests is largely underestimated, but also that many lineages are millions of years old. The genera Phyzelaphryne (1 sp.) and Adelophryne (6 spp.), which compose the subfamily Phyzelaphryninae, include poorly documented, secretive, and minute frogs with an unusual distribution pattern that encompasses the biotic disjunction between Amazonia and the Atlantic forest. We generated >5.8 kb sequence data from six markers for all seven nominal species of the subfamily as well as for newly discovered populations in order to (1) test the monophyly of Phyzelaphryninae, Adelophryne and Phyzelaphryne, (2) estimate species diversity within the subfamily, and (3) investigate their historical biogeography and diversification. Phylogenetic reconstruction confirmed the monophyly of each group and revealed deep subdivisions within Adelophryne and Phyzelaphryne, with three major clades in Adelophryne located in northern Amazonia, northern Atlantic forest and southern Atlantic forest. Our results suggest that the actual number of species in Phyzelaphryninae is, at least, twice the currently recognized species diversity, with almost every geographically isolated population representing an anciently divergent candidate species. Such results highlight the challenges for conservation, especially in the northern Atlantic forest where it is still degraded at a fast pace. Molecular dating revealed that Phyzelaphryninae originated in Amazonia and dispersed during early Miocene to the Atlantic forest. The two Atlantic forest clades of Adelophryne started to diversify some 7 Ma minimum, while the northern Amazonian Adelophryne diversified much earlier, some 13 Ma minimum. This striking biogeographic pattern coincides with

  12. Genome-wide expression profiling in the Drosophila eye reveals unexpected repression of Notch signaling by the JAK/STAT pathway

    PubMed Central

    Flaherty, Maria Sol; Zavadil, Jiri; Ekas, Laura A.; Bach, Erika A.

    2010-01-01

    Although the JAK/STAT pathway regulates numerous processes in vertebrates and invertebrates through modulating transcription, its functionally-relevant transcriptional targets remain largely unknown. With one jak and one stat (stat92E), Drosophila provides a powerful system for finding new JAK/STAT target genes. Genome-wide expression profiling on eye discs in which Stat92E is hyperactivated, revealed 584 differentially-regulated genes, including known targets domeless, socs36E and wingless. Other differentially-regulated genes (chinmo, lama, Mo25, Imp-L2, Serrate, Delta) were validated and may represent new Stat92E targets. Genetic experiments revealed that Stat92E cell-autonomously represses Serrate, which encodes a Notch ligand. Loss of Stat92E led to de-repression of Serrate in the dorsal eye, resulting in ectopic Notch signaling and aberrant eye growth there. Thus, our micro-array documents a new Stat92E target gene and a previously-unidentified inhibitory action of Stat92E on Notch signaling. These data suggest that this study will be a useful resource for the identification of additional Stat92E targets. PMID:19504457

  13. Genome-wide expression profiling in the Drosophila eye reveals unexpected repression of notch signaling by the JAK/STAT pathway.

    PubMed

    Flaherty, Maria Sol; Zavadil, Jiri; Ekas, Laura A; Bach, Erika A

    2009-09-01

    Although the JAK/STAT pathway regulates numerous processes in vertebrates and invertebrates through modulating transcription, its functionally relevant transcriptional targets remain largely unknown. With one jak and one stat (stat92E), Drosophila provides a powerful system for finding new JAK/STAT target genes. Genome-wide expression profiling on eye discs in which Stat92E is hyperactivated, revealed 584 differentially regulated genes, including known targets domeless, socs36E, and wingless. Other differentially regulated genes (chinmo, lama, Mo25, Imp-L2, Serrate, Delta) were validated and may represent new Stat92E targets. Genetic experiments revealed that Stat92E cell-autonomously represses Serrate, which encodes a Notch ligand. Loss of Stat92E led to de-repression of Serrate in the dorsal eye, resulting in ectopic Notch signaling and aberrant eye growth there. Thus, our micro-array documents a new Stat92E target gene and a previously unidentified inhibitory action of Stat92E on Notch signaling. These data suggest that this study will be a useful resource for the identification of additional Stat92E targets. PMID:19504457

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

    PubMed

    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. PMID:26058515

  15. Revealing the complex conduction heat transfer mechanism of nanofluids

    NASA Astrophysics Data System (ADS)

    Sergis, A.; Hardalupas, Y.

    2015-06-01

    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

  16. Layered Social Network Analysis Reveals Complex Relationships in Kindergarteners.

    PubMed

    Golemiec, Mireille; Schneider, Jonathan; Boyce, W Thomas; Bush, Nicole R; Adler, Nancy; Levine, Joel D

    2016-01-01

    The interplay between individuals forms building blocks for social structure. Here, we examine the structure of behavioral interactions among kindergarten classroom with a hierarchy-neutral approach to examine all possible underlying patterns in the formation of layered networks of "reciprocal" interactions. To understand how these layers are coordinated, we used a layered motif approach. Our dual layered motif analysis can therefore be thought of as the dynamics of smaller groups that tile to create the group structure, or alternatively they provide information on what the average child would do in a given local social environment. When we examine the regulated motifs in layered networks, we find that transitivity is at least partially involved in the formation of these layered network structures. We also found complex combinations of the expected reciprocal interactions. The mechanisms used to understand social networks of kindergarten children here are also applicable on a more general scale to any group of individuals where interactions and identities can be readily observed and scored. PMID:26973572

  17. Layered Social Network Analysis Reveals Complex Relationships in Kindergarteners

    PubMed Central

    Golemiec, Mireille; Schneider, Jonathan; Boyce, W. Thomas; Bush, Nicole R.; Adler, Nancy; Levine, Joel D.

    2016-01-01

    The interplay between individuals forms building blocks for social structure. Here, we examine the structure of behavioral interactions among kindergarten classroom with a hierarchy-neutral approach to examine all possible underlying patterns in the formation of layered networks of “reciprocal” interactions. To understand how these layers are coordinated, we used a layered motif approach. Our dual layered motif analysis can therefore be thought of as the dynamics of smaller groups that tile to create the group structure, or alternatively they provide information on what the average child would do in a given local social environment. When we examine the regulated motifs in layered networks, we find that transitivity is at least partially involved in the formation of these layered network structures. We also found complex combinations of the expected reciprocal interactions. The mechanisms used to understand social networks of kindergarten children here are also applicable on a more general scale to any group of individuals where interactions and identities can be readily observed and scored. PMID:26973572

  18. Laser altimetry reveals complex pattern of Greenland Ice Sheet dynamics.

    PubMed

    Csatho, Beata M; Schenk, Anton F; van der Veen, Cornelis J; Babonis, Gregory; Duncan, Kyle; Rezvanbehbahani, Soroush; van den Broeke, Michiel R; Simonsen, Sebastian B; Nagarajan, Sudhagar; van Angelen, Jan H

    2014-12-30

    We present a new record of ice thickness change, reconstructed at nearly 100,000 sites on the Greenland Ice Sheet (GrIS) from laser altimetry measurements spanning the period 1993-2012, partitioned into changes due to surface mass balance (SMB) and ice dynamics. We estimate a mean annual GrIS mass loss of 243 ± 18 Gt ⋅ y(-1), equivalent to 0.68 mm ⋅ y(-1) sea level rise (SLR) for 2003-2009. Dynamic thinning contributed 48%, with the largest rates occurring in 2004-2006, followed by a gradual decrease balanced by accelerating SMB loss. The spatial pattern of dynamic mass loss changed over this time as dynamic thinning rapidly decreased in southeast Greenland but slowly increased in the southwest, north, and northeast regions. Most outlet glaciers have been thinning during the last two decades, interrupted by episodes of decreasing thinning or even thickening. Dynamics of the major outlet glaciers dominated the mass loss from larger drainage basins, and simultaneous changes over distances up to 500 km are detected, indicating climate control. However, the intricate spatiotemporal pattern of dynamic thickness change suggests that, regardless of the forcing responsible for initial glacier acceleration and thinning, the response of individual glaciers is modulated by local conditions. Recent projections of dynamic contributions from the entire GrIS to SLR have been based on the extrapolation of four major outlet glaciers. Considering the observed complexity, we question how well these four glaciers represent all of Greenland's outlet glaciers. PMID:25512537

  19. Fractal analysis reveals reduced complexity of retinal vessels in CADASIL.

    PubMed

    Cavallari, Michele; Falco, Teresa; Frontali, Marina; Romano, Silvia; Bagnato, Francesca; Orzi, Francesco

    2011-01-01

    The Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) affects mainly small cerebral arteries and leads to disability and dementia. The relationship between clinical expression of the disease and progression of the microvessel pathology is, however, uncertain as we lack tools for imaging brain vessels in vivo. Ophthalmoscopy is regarded as a window into the cerebral microcirculation. In this study we carried out an ophthalmoscopic examination in subjects with CADASIL. Specifically, we performed fractal analysis of digital retinal photographs. Data are expressed as mean fractal dimension (mean-D), a parameter that reflects complexity of the retinal vessel branching. Ten subjects with genetically confirmed diagnosis of CADASIL and 10 sex and age-matched control subjects were enrolled. Fractal analysis of retinal digital images was performed by means of a computer-based program, and the data expressed as mean-D. Brain MRI lesion volume in FLAIR and T1-weighted images was assessed using MIPAV software. Paired t-test was used to disclose differences in mean-D between CADASIL and control groups. Spearman rank analysis was performed to evaluate potential associations between mean-D values and both disease duration and disease severity, the latter expressed as brain MRI lesion volumes, in the subjects with CADASIL. The results showed that mean-D value of patients (1.42±0.05; mean±SD) was lower than control (1.50±0.04; p = 0.002). Mean-D did not correlate with disease duration nor with MRI lesion volumes of the subjects with CADASIL. The findings suggest that fractal analysis is a sensitive tool to assess changes of retinal vessel branching, likely reflecting early brain microvessel alterations, in CADASIL patients. PMID:21556373

  20. Measurement of PIP3 Levels Reveals an Unexpected Role for p110β in Early Adaptive Responses to p110α-Specific Inhibitors in Luminal Breast Cancer

    PubMed Central

    Costa, Carlotta; Ebi, Hiromichi; Martini, Miriam; Beausoleil, Sean A.; Faber, Anthony C.; Jakubik, Charles T.; Huang, Alan; Wang, Youzhen; Nishtala, Madhuri; Hall, Ben; Rikova, Klarisa; Zhao, Jean; Hirsch, Emilio; Benes, Cyril H.

    2016-01-01

    SUMMARY BYL719, which selectively inhibits the alpha isoform of the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110α), is currently in clinical trials for the treatment of solid tumors, especially luminal breast cancers with PIK3CA mutations and/or HER2 amplification. This study reveals that, even among these sensitive cancers, the initial efficacy of p110α inhibition is mitigated by rapid re-accumulation of the PI3K product PIP3 produced by the p110β isoform. Importantly, the reactivation of PI3K mediated by p110β does not invariably restore AKT phosphorylation, demonstrating the limitations of using phospho-AKT as a surrogate to measure PI3K activation. Consistently, we show that the addition of the p110β inhibitor to BYL719 prevents the PIP3 rebound and induces greater antitumor efficacy in HER2-amplified and PIK3CA mutant cancers. PMID:25544637

  1. NMR structures of two variants of bovine pancreatic trypsin inhibitor (BPTI) reveal unexpected influence of mutations on protein structure and stability.

    PubMed

    Cierpicki, Tomasz; Otlewski, Jacek

    2002-08-23

    Here we determined NMR solution structures of two mutants of bovine pancreatic trypsin inhibitor (BPTI) to reveal structural reasons of their decreased thermodynamic stability. A point mutation, A16V, in the solvent-exposed loop destabilizes the protein by 20 degrees C, in contrast to marginal destabilization observed for G, S, R, L or W mutants. In the second mutant introduction of eight alanine residues at proteinase-contacting sites (residues 11, 13, 17, 18, 19, 34, 37 and 39) provides a protein that denatures at a temperature about 30 degrees C higher than expected from additive behavior of individual mutations. In order to efficiently determine structures of these variants, we applied a procedure that allows us to share data between regions unaffected by mutation(s). NOAH/DYANA and CNS programs were used for a rapid assignment of NOESY cross-peaks, structure calculations and refinement. The solution structure of the A16V mutant reveals no conformational change within the molecule, but shows close contacts between V16, I18 and G36/G37. Thus, the observed 4.3kcal/mol decrease of stability results from a strained local conformation of these residues caused by introduction of a beta-branched Val side-chain. Contrary to the A16V mutation, introduction of eight alanine residues produces significant conformational changes, manifested in over a 9A shift of the Y35 side-chain. This structural rearrangement provides about 6kcal/mol non-additive stabilization energy, compared to the mutant in which G37 and R39 are not mutated to alanine residues. PMID:12206780

  2. Severe Left Ventricular Hypertrophy, Small Pericardial Effusion, and Diffuse Late Gadolinium Enhancement by Cardiac Magnetic Resonance Suspecting Cardiac Amyloidosis: Endomyocardial Biopsy Reveals an Unexpected Diagnosis

    PubMed Central

    Hofmann, Nina P.; Giusca, Sorin; Klingel, Karin; Nunninger, Peter; Korosoglou, Grigorios

    2016-01-01

    Left ventricular (LV) hypertrophy can be related to a multitude of cardiac disorders, such as hypertrophic cardiomyopathy (HCM), cardiac amyloidosis, and hypertensive heart disease. Although the presence of LV hypertrophy is generally associated with poorer cardiac outcomes, the early differentiation between these pathologies is crucial due to the presence of specific treatment options. The diagnostic process with LV hypertrophy requires the integration of clinical evaluation, electrocardiography (ECG), echocardiography, biochemical markers, and if required CMR and endomyocardial biopsy in order to reach the correct diagnosis. Here, we present a case of a patient with severe LV hypertrophy (septal wall thickness of 23 mm, LV mass of 264 g, and LV mass index of 147 g/m2), severely impaired longitudinal function, and preserved radial contractility (ejection fraction = 55%), accompanied by small pericardial effusion and diffuse late gadolinium enhancement (LGE) by cardiac magnetic resonance (CMR). Due to the imaging findings, an infiltrative cardiomyopathy, such as cardiac amyloidosis, was suspected. However, amyloid accumulation was excluded by endomyocardial biopsy, which revealed the presence of diffuse myocardial fibrosis in an advanced hypertensive heart disease. PMID:27247807

  3. The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation

    SciTech Connect

    Hansen, Guido; Hercus, Timothy R.; McClure, Barbara J.; Stomski, Frank C.; Dottore, Mara; Powell, Jason; Ramshaw, Hayley; Woodcock, Joanna M.; Xu, Yibin; Guthridge, Mark; McKinstry, William J.; Lopez, Angel F.; Parker, Michael W.

    2008-08-11

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific {alpha} subunit and a {beta}c subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

  4. The structure of the follistatin:activin complex reveals antagonism of both type I and type II receptor binding

    SciTech Connect

    Thompson, T.B.; Lerch, T.F.; Cook, R.W.; Woodruff, T.K.; Jardetzky, T.S.

    2010-03-08

    TGF-{beta} ligands stimulate diverse cellular differentiation and growth responses by signaling through type I and II receptors. Ligand antagonists, such as follistatin, block signaling and are essential regulators of physiological responses. Here we report the structure of activin A, a TGF-{beta} ligand, bound to the high-affinity antagonist follistatin. Two follistatin molecules encircle activin, neutralizing the ligand by burying one-third of its residues and its receptor binding sites. Previous studies have suggested that type I receptor binding would not be blocked by follistatin, but the crystal structure reveals that the follistatin N-terminal domain has an unexpected fold that mimics a universal type I receptor motif and occupies this receptor binding site. The formation of follistatin:BMP:type I receptor complexes can be explained by the stoichiometric and geometric arrangement of the activin:follistatin complex. The mode of ligand binding by follistatin has important implications for its ability to neutralize homo- and heterodimeric ligands of this growth factor family.

  5. Effectiveness of Annealing Blocking Primers versus Restriction Enzymes for Characterization of Generalist Diets: Unexpected Prey Revealed in the Gut Contents of Two Coral Reef Fish Species

    PubMed Central

    Leray, Matthieu; Agudelo, Natalia; Mills, Suzanne C.; Meyer, Christopher P.

    2013-01-01

    Characterization of predator-prey interactions is challenging as researchers have to rely on indirect methods that can be costly, biased and too imprecise to elucidate the complexity of food webs. DNA amplification and sequencing techniques of gut and fecal contents are promising approaches, but their success largely depends on the ability to amplify the taxonomic array of prey consumed and then match prey amplicons with reference sequences. When little a priori information on diet is available or a generalist predator is targeted, versatile primer sets (also referred to as universal or general primers) as opposed to group- or species-specific primer sets are the most powerful to unveil the full range of prey consumed. However, versatile primers are likely to preferentially amplify the predominant, less degraded predator DNA if no manipulation is performed to exclude this confounding DNA template. In this study we compare two approaches that eliminate the confounding predator template: restriction digestion and the use of annealing blocking primers. First, we use a preliminary DNA barcode library provided by the Moorea BIOCODE project to 1) evaluate the cutting frequency of commercially available restriction enzymes and 2) design predator specific annealing blocking primers. We then compare the performance of the two predator removal strategies for the detection of prey templates using two versatile primer sets from the gut contents of two generalist coral reef fish species sampled in Moorea. Our study demonstrates that blocking primers should be preferentially used over restriction digestion for predator DNA removal as they recover greater prey diversity. We also emphasize that a combination of versatile primers may be required to best represent the breadth of a generalist's diet. PMID:23579925

  6. In vivo Whole-Cell Recordings Combined with Electron Microscopy Reveal Unexpected Morphological and Physiological Properties in the Lateral Nucleus of the Trapezoid Body in the Auditory Brainstem

    PubMed Central

    Franken, Tom P.; Smith, Philip H.; Joris, Philip X.

    2016-01-01

    The lateral nucleus of the trapezoid body (LNTB) is a prominent nucleus in the superior olivary complex in mammals including humans. Its physiology in vivo is poorly understood due to a paucity of recordings. It is thought to provide a glycinergic projection to the medial superior olive (MSO) with an important role in binaural processing and sound localization. We combined in vivo patch clamp recordings with labeling of individual neurons in the Mongolian gerbil. Labeling of the recorded neurons allowed us to relate physiological properties to anatomy at the light and electron microscopic level. We identified a population of quite dorsally located neurons with surprisingly large dendritic trees on which most of the synaptic input impinges. In most neurons, one or more of these dendrites run through and are then medial to the MSO. These neurons were often binaural and could even show sensitivity to interaural time differences (ITDs) of stimulus fine structure or envelope. Moreover, a subpopulation showed enhanced phase-locking to tones delivered in the tuning curve tail. We propose that these neurons constitute the gerbil main LNTB (mLNTB). In contrast, a smaller sample of neurons was identified that was located more ventrally and that we designate to be in posteroventral LNTB (pvLNTB). These cells receive large somatic excitatory terminals from globular bushy cells. We also identified previously undescribed synaptic inputs from the lateral superior olive. pvLNTB neurons are usually monaural, display a primary-like-with-notch response to ipsilateral short tones at CF and can phase-lock to low frequency tones. We conclude that mLNTB contains a population of neurons with extended dendritic trees where most of the synaptic input is found, that can show enhanced phase-locking and sensitivity to ITD. pvLNTB cells, presumed to provide glycinergic input to the MSO, get large somatic globular bushy synaptic inputs and are typically monaural with short tone responses similar

  7. In vivo Whole-Cell Recordings Combined with Electron Microscopy Reveal Unexpected Morphological and Physiological Properties in the Lateral Nucleus of the Trapezoid Body in the Auditory Brainstem.

    PubMed

    Franken, Tom P; Smith, Philip H; Joris, Philip X

    2016-01-01

    The lateral nucleus of the trapezoid body (LNTB) is a prominent nucleus in the superior olivary complex in mammals including humans. Its physiology in vivo is poorly understood due to a paucity of recordings. It is thought to provide a glycinergic projection to the medial superior olive (MSO) with an important role in binaural processing and sound localization. We combined in vivo patch clamp recordings with labeling of individual neurons in the Mongolian gerbil. Labeling of the recorded neurons allowed us to relate physiological properties to anatomy at the light and electron microscopic level. We identified a population of quite dorsally located neurons with surprisingly large dendritic trees on which most of the synaptic input impinges. In most neurons, one or more of these dendrites run through and are then medial to the MSO. These neurons were often binaural and could even show sensitivity to interaural time differences (ITDs) of stimulus fine structure or envelope. Moreover, a subpopulation showed enhanced phase-locking to tones delivered in the tuning curve tail. We propose that these neurons constitute the gerbil main LNTB (mLNTB). In contrast, a smaller sample of neurons was identified that was located more ventrally and that we designate to be in posteroventral LNTB (pvLNTB). These cells receive large somatic excitatory terminals from globular bushy cells. We also identified previously undescribed synaptic inputs from the lateral superior olive. pvLNTB neurons are usually monaural, display a primary-like-with-notch response to ipsilateral short tones at CF and can phase-lock to low frequency tones. We conclude that mLNTB contains a population of neurons with extended dendritic trees where most of the synaptic input is found, that can show enhanced phase-locking and sensitivity to ITD. pvLNTB cells, presumed to provide glycinergic input to the MSO, get large somatic globular bushy synaptic inputs and are typically monaural with short tone responses similar

  8. [Unexpected drug-interaction].

    PubMed

    Tajima, Yutaka

    2002-02-01

    The case of a male patient suffering from chronic normal pressure hydrocephalus is outlined. Antidepressant and pravastatin were administered because of the patient's abulia and hypercholesterolemia, but neuroleptic malignant syndrome-like conditions developed. All physicians should suppose the occurrence of such an "unexpected drug-interaction" in any case. The author considered that a good sense of careful discernment and rapid reference system of medical information are "essential tools" for clinical management. PMID:11925849

  9. Revealing Classroom Complexity: A Portrait of a Justice-Oriented, Democratic Curriculum Serving a Disadvantaged Neighborhood

    ERIC Educational Resources Information Center

    Schultz, Brian D.

    2006-01-01

    This study discusses my attempt to improve educational experiences of fifth-grade students living in public housing. Through reconstruction of my thought processes while teaching and learning with students, the context of social justice-oriented teaching and classroom complexity is revealed. A narrative portrayal emerges demonstrating the impact…

  10. Increasing phonological complexity reveals heightened instability in inter-articulatory coordination in adults who stutter

    PubMed Central

    Smith, Anne; Sadagopan, Neeraja; Walsh, Bridget; Weber-Fox, Christine

    2010-01-01

    The potential role of phonological complexity in destabilizing the speech motor systems of adults who stutter was explored by assessing the performance of 17 adults who stutter and 17 matched control participants on a nonword repetition task. The nonwords varied in length and phonological complexity. Behavioral results revealed no differences between the stuttering and normally fluent groups on accuracy of nonword repetition. In contrast, dramatic differences between groups were observed in the kinematic data. Indices of the consistency of inter-articulator coordination revealed that adults who stutter were much less consistent in their coordinative patterns over repeated productions. With increasing length and complexity of the nonwords, between-group differences in coordinative consistency were more pronounced. Coordination consistency measures revealed that adults who stutter (but not normally fluent adults) showed within-session practice effects; their coordinative consistency improved in five later compared to five earlier productions. Adults who stutter produced the nonwords at a slower rate, but both groups showed increased rates of production on the later trials, indicating a practice effect for duration for both groups. We conclude that, though the adults who stutter performed behaviorally with the same accuracy as normally fluent adults, the nonword repetition task reveals remarkable differences in the speech motor dynamics underlying fluent speech production in adults who stutter compared to their normally fluent peers. These results support a multifactorial, dynamic model of stuttering in which linguistic complexity and utterance length are factors that contribute to the probability of breakdown of the speech motor system. PMID:20412979

  11. Combining complexity measures of EEG data: multiplying measures reveal previously hidden information

    PubMed Central

    Burns, Thomas; Rajan, Ramesh

    2015-01-01

    Many studies have noted significant differences among human electroencephalograph (EEG) results when participants or patients are exposed to different stimuli, undertaking different tasks, or being affected by conditions such as epilepsy or Alzheimer's disease. Such studies often use only one or two measures of complexity and do not regularly justify their choice of measure beyond the fact that it has been used in previous studies. If more measures were added to such studies, however, more complete information might be found about these reported differences. Such information might be useful in confirming the existence or extent of such differences, or in understanding their physiological bases. In this study we analysed publically-available EEG data using a range of complexity measures to determine how well the measures correlated with one another. The complexity measures did not all significantly correlate, suggesting that different measures were measuring unique features of the EEG signals and thus revealing information which other measures were unable to detect. Therefore, the results from this analysis suggests that combinations of complexity measures reveal unique information which is in addition to the information captured by other measures of complexity in EEG data. For this reason, researchers using individual complexity measures for EEG data should consider using combinations of measures to more completely account for any differences they observe and to ensure the robustness of any relationships identified. PMID:26594331

  12. Combining complexity measures of EEG data: multiplying measures reveal previously hidden information.

    PubMed

    Burns, Thomas; Rajan, Ramesh

    2015-01-01

    Many studies have noted significant differences among human electroencephalograph (EEG) results when participants or patients are exposed to different stimuli, undertaking different tasks, or being affected by conditions such as epilepsy or Alzheimer's disease. Such studies often use only one or two measures of complexity and do not regularly justify their choice of measure beyond the fact that it has been used in previous studies. If more measures were added to such studies, however, more complete information might be found about these reported differences. Such information might be useful in confirming the existence or extent of such differences, or in understanding their physiological bases. In this study we analysed publically-available EEG data using a range of complexity measures to determine how well the measures correlated with one another. The complexity measures did not all significantly correlate, suggesting that different measures were measuring unique features of the EEG signals and thus revealing information which other measures were unable to detect. Therefore, the results from this analysis suggests that combinations of complexity measures reveal unique information which is in addition to the information captured by other measures of complexity in EEG data. For this reason, researchers using individual complexity measures for EEG data should consider using combinations of measures to more completely account for any differences they observe and to ensure the robustness of any relationships identified. PMID:26594331

  13. Reticulate evolution of the Daphnia pulex complex as revealed by nuclear markers.

    PubMed

    Vergilino, Roland; Markova, Silvia; Ventura, Marc; Manca, Marina; Dufresne, France

    2011-03-01

    The study of species complexes is of particular interest to understand how evolutionary young species maintain genomic integrity. The Daphnia pulex complex has been intensively studied as it includes species that dominate freshwater environments in the Northern hemisphere and as it is the sole North American complex that shows transitions to obligate parthenogenesis. Past studies using mitochondrial markers have revealed the presence of 10 distinct lineages in the complex. This study is the first to examine genetic relationships among seven species of the complex at nuclear markers (nine microsatellite loci and one protein-coding gene). Clones belonging to the seven species of the Daphnia pulex complex were characterized at the mitochondrial NADH dehydrogenase (ND5) gene and at the Lactate dehydrogenase (LDH) locus. K-means, principal coordinate analyses and phylogenetic network analyses on the microsatellite data all separated European D. pulicaria, D. tenebrosa, North American D. pulex, D. pulicaria and their hybrids into distinct clusters. The hybrid cluster was composed of diploid and polyploid hybrids with D. pulex mitochondria and some clones with D. pulicaria mitochondria. By contrast, the phylogeny of the D. pulex complex using Rab4 was not well resolved but still showed clusters consisting mostly of D. pulex alleles and others of D. pulicaria alleles. Incomplete lineage sorting and hybridization may obscure genetic relationships at this locus. This study shows that hybridization and introgression have played an important role in the evolution of this complex. PMID:21294799

  14. Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes.

    PubMed

    Dean, Samuel; Moreira-Leite, Flavia; Varga, Vladimir; Gull, Keith

    2016-08-30

    The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics. PMID:27519801

  15. Unexpected high photothemal conversion efficiency of gold nanospheres upon grafting with two-photon luminescent ruthenium(II) complexes: A way towards cancer therapy?

    PubMed

    Zhang, Pingyu; Wang, Jinquan; Huang, Huaiyi; Yu, Bole; Qiu, Kangqiang; Huang, Juanjuan; Wang, Shutao; Jiang, Lei; Gasser, Gilles; Ji, Liangnian; Chao, Hui

    2015-09-01

    The design and development of functional hybrid nanomaterials is currently a topic of great interest in biomedicine. Herein we investigated the grafting of Ru(II) polypyridyl complexes onto gold nanospheres (Ru@AuNPs) to improve the particles' near infrared (NIR) absorption, and ultimately allow for application in photothermal cancer therapy. As demonstrated in this article, these ruthenium(II) complexes could indeed significantly enhance gold nanospheres' two-photon luminescence (PTL) intensity and photothermal therapy (PTT) efficiency. The best dual functional nanoparticles of this study were successfully used for real-time luminescent imaging-guided PTT in live cancer cells. Furthermore, in vivo tumor ablation was achieved with excellent treatment efficacy under a diode laser (808 nm) irradiation at the power density of 0.8 W/cm(2) for 5 min. This study demonstrates that the coupling of inert Ru(II) polypyridyl complexes to gold nanospheres allows for the enhancement of two-photon luminescence and for efficient photothermal effect. PMID:26093791

  16. Proteomic Insight Reveals Elevated Levels of Albumin in Circulating Immune Complexes in Diabetic Plasma.

    PubMed

    Bhat, Shweta; Jagadeeshaprasad, Mashanipalya G; Patil, Yugendra R; Shaikh, Mahemud L; Regin, Bhaskaran S; Mohan, Viswanathan; Giri, Ashok P; Balasubramanyam, Muthuswamy; Boppana, Ramanamurthy; Kulkarni, Mahesh J

    2016-06-01

    A Hyperglycemic condition in diabetes promotes formation of advanced glycation end products, which are known to elicit immune response and form complexes with immunoglobulins called circulating immune complexes. To investigate the involvement of advanced glycation end product (AGE)-modified proteins in the elicitation of an immune response, circulating immune complexes were isolated and proteins associated were identified and characterized. Label-free-based mass spectrometric analysis of circulating immune complexes in clinical plasma of prediabetic, newly diagnosed diabetes, and diabetic microalbuminurea revealed elevated levels of serum albumin in the circulating immune complexes, which were also observed to be AGE modified. Further, to examine the role of glycation, circulating immune complexeswere analyzed in the streptozotocin-induced diabetic mice treated with or without aminoguanidine, a prototype glycation inhibitor. Mass spectrometric analysis of circulating immune complexes showed elevated levels of serum albumin in plasma from diabetic mice over that of control animals. Aminoguanidine-treated diabetic mice displayed decreased AGE modification of plasma albumin, accompanied by a reduced level of albumin in the circulating immune complexes. In addition, elevated levels of proinflammatory cytokines such as IL-1b, IL-2, and TNF-alpha were observed in diabetes, which were reduced with aminoguanidine treatment, suggesting the involvement of glycation in the immune response. PMID:27056913

  17. On the way of revealing coactivator complexes cross-talk during transcriptional activation.

    PubMed

    Krasnov, Aleksey N; Mazina, Marina Yu; Nikolenko, Julia V; Vorobyeva, Nadezhda E

    2016-01-01

    Transcriptional activation is a complex, multistage process implemented by hundreds of proteins. Many transcriptional proteins are organized into coactivator complexes, which participate in transcription regulation at numerous genes and are a driver of this process. The molecular action mechanisms of coactivator complexes remain largely understudied. Relevant publications usually deal with the involvement of these complexes in the entire process of transcription, and only a few studies are aimed to elucidate their functions at its particular stages. This review summarizes available information on the participation of key coactivator complexes in transcriptional activation. The timing of coactivator complex binding/removal has been used for restructuring previously described information about the transcriptional process. Several major stages of transcriptional activation have been distinguished based on the presence of covalent histone modifications and general transcriptional factors, and the recruitment and/or removal phases have been determined for each coactivator included in analysis. Recruitment of Mediator, SWItch/Sucrose Non-Fermentable and NUcleosome Remodeling Factor complexes during transcription activation has been investigated thoroughly; CHD and INOsitol auxotrophy 80 families are less well studied. In most cases, the molecular mechanisms responsible for the removal of certain coactivator complexes after the termination of their functions at the promoters are still not understood. On the basis of the summarized information, we propose a scheme that illustrates the involvement of coactivator complexes in different stages of the transcription activation process. This scheme may help to gain a deeper insight into the molecular mechanism of functioning of coactivator complexes, find novel participants of the process, and reveal novel structural or functional connections between different coactivators. PMID:26913181

  18. Structural and magnetic modulations of copper(II) azido complexes: unexpected in situ reactions of mono-N-donor pyridine-based co-ligands.

    PubMed

    Zhao, Jiong-Peng; Xie, Yabo; Li, Jian-Rong; Bu, Xian-He

    2016-01-28

    Low-temperature hydrothermal reactions of copper salts with NaN3 and structurally related pyridine-based ligands, 1-(4-pyridyl)pyridinium, 3-chloromethylpyridine, 4-benzylpyridine, and quinoline (L(4)), respectively, led to the formation of four new magnetic complexes, [Cu3(L(1))2(N3)6]n (), [Cu3(L(2))2(N3)6]n (), [Cu(L(3))2(N3)2] (), and [Cu(L(4))(N3)2]n (). In these complexes, L(1), L(2), and L(3) are pyridine, 3-azidomethylpyridine, and 4-benzoylpyridine, being generated in situ by the decomposition, azido substitution, and oxidation reaction of the 1-(4-pyridyl)pyridinium, 3-chloromethylpyridine, and 4-benzylpyridine, respectively. and have similar structures being composed of double end-on azido-bridging planar [Cu3(L)2(N3)6] trinuclear units, which are further linked into a two-dimensional layer by the end-to-end azido bridges of themselves, along with their weak end-on coordination. It is interesting that the only slight differences of geometrical parameters in and have led to distinct magnetic interactions between the trinuclear units, where the former is antiferromagnetic but the latter is ferromagnetic, whereas has a mono-nuclear core structure, which is further extended to a one-dimensional (1D) chain by weakly coordinated end-on azido bridges. consists of unique 1D chains with double end-on azido bridge bonding distorted five-coordinated Cu(ii) centers, and exhibits ferromagnetic intrachain interactions. In the structures of these complexes there also exist weak inter-layer or inter-chain hydrogen bonds, which should also be responsible for some magnetic behavior at low temperature. In addition, primary structural and magnetic comparisons and discussions have also been performed by combining other reported azido-Cu(ii) systems with related pyridyl-based co-ligands. These results show that the selection of synthesis conditions and slight decoration of co-ligands (or geometric differences of them) have important influences on the structures and magnetic

  19. Unexpected formation of a fused double cycle trinuclear gold(i) complex supported by ortho-phenyl metallated aryl-diphosphine ligands and strong aurophilic interactions.

    PubMed

    Jobbágy, Csaba; Baranyai, Péter; Szabó, Pál; Holczbauer, Tamás; Rácz, Barbara; Li, Liang; Naumov, Panče; Deák, Andrea

    2016-08-01

    The first homoleptic trinuclear arylgold(i) complex, [Au3(L')2](NO3) (3), based on an ortho-phenyl metallated aryl-diphosphine ligand (L' = o-C6H4PPh(C15H10O)PPh2), has been obtained through a new thermolytic reaction of the corresponding diauracycle, [Au2(L)2](NO3)2 (L = xantphos). The formation of 3 involves activation of the ortho-phenyl C-H bond of the xantphos ligands. The presence of Au-C bonds in this new gold-diphosphine cluster is not its only remarkable feature, since it also displays two 12-membered rings fused together and a linear {Au3} chain with aurophilic interactions. Complex 3 exhibits strong sky-blue luminescence that can be assigned to a triplet metal-metal ((3)MM) transition partially mixed with a ligand-to-metal-metal charge transfer ((3)LMMCT) transition related to the aurophilic bonding. This [Au3(L')2](+) triauracycle also shows AIEE-activity, and is a selective luminescent chemosensor for metal ions. PMID:27439467

  20. Insight into the dynamics of lanthanide-DTPA complexes as revealed by oxygen-17 NMR.

    PubMed

    Fusaro, Luca; Mocci, Francesca; Muller, Robert N; Luhmer, Michel

    2012-08-01

    DTPA chelates of various diamagnetic and paramagnetic lanthanide(III) metal ions, as well as the chemically similar DTPA chelate of Y(3+), were studied in aqueous solution by variable temperature (17)O NMR with the aim of characterizing their internal dynamics. As a consequence of poor chemical shift dispersion and fast quadrupole relaxation, no dynamic exchange process could be detected for the diamagnetic complexes nor for the Sm-DTPA complex. In contrast, the spectra recorded for the Eu-DTPA complex show chemical exchange due to the well-known racemization process and, at high temperature, feature signal broadening that reveals a fluxional process involving the interchange of the coordinated and noncoordinated oxygen atoms of the carboxylate groups. The spectra recorded for the Pr-DTPA complex feature coalescence events due to such a fluxional process, which is ascribable to the rotation of the carboxylate groups. The activation free energy barriers determined experimentally are remarkably lower than the calculated activation barriers recently reported for the rotation of the carboxylate groups of various Ln-DOTA complexes. Furthermore, the smallest activation free energy measured for the Pr-DTPA complex, about 45 kJ mol(-1), is significantly lower than the activation free energy characterizing the racemization process. The fluxional behavior of the carboxylate groups is, however, not expected to significantly affect the residence time of the water molecule coordinated to the metal ion. PMID:22817329

  1. Deep Neural Networks Reveal a Gradient in the Complexity of Neural Representations across the Ventral Stream.

    PubMed

    Güçlü, Umut; van Gerven, Marcel A J

    2015-07-01

    Converging evidence suggests that the primate ventral visual pathway encodes increasingly complex stimulus features in downstream areas. We quantitatively show that there indeed exists an explicit gradient for feature complexity in the ventral pathway of the human brain. This was achieved by mapping thousands of stimulus features of increasing complexity across the cortical sheet using a deep neural network. Our approach also revealed a fine-grained functional specialization of downstream areas of the ventral stream. Furthermore, it allowed decoding of representations from human brain activity at an unsurpassed degree of accuracy, confirming the quality of the developed approach. Stimulus features that successfully explained neural responses indicate that population receptive fields were explicitly tuned for object categorization. This provides strong support for the hypothesis that object categorization is a guiding principle in the functional organization of the primate ventral stream. PMID:26157000

  2. An unexpected effect of an ouabain-sensitive ATPase activity on the amount of antigen-antibody complexes formed in situ.

    PubMed

    Mentré, P; Debey, P

    1999-09-01

    A classical method of indirect immunofluorescence was applied on various kinds of lightly fixed and permeabilized cells to analyze the formation of the complexes between a nuclear antigen and its antibody (AAC). The amount of AAC decreased dramatically when the incubation with the first antibody was realized in the presence of ATP in a sodium-rich medium with 0.5 mM KCl. Addition of sodium vanadate, a general inhibitor of ATPases, ouabain or tetrabutylammonium ion, specific inhibitors of the Na+,K+-ATPase, prevented this effect. The established role of this enzyme is to increase free-K+ concentration and decrease free Na+ concentration in the cell. It is not surprising to find an ATPase still active since light fixation and permeabilization do not destroy phosphatases. But it is rather surprising to find something looking like Na+,K+-ATPase activity in permeabilized cells. The importance of potassium in this puzzling result is suggested by the fact that appearance of ACC was equally suppressed if the incubation was made in the absence of ATP in a potassium-rich medium without sodium. Results are discussed, taking into account the properties of cell-associated water and recently found interrelation between Na+,K+-ATPase and tubulin. In any case, the results seem interesting in the field of immunocytochemistry. PMID:10541475

  3. Modelling Coral Biomineralization: Mixed Kinetic/Equilibrium Trace Element Coprecipitation Models Reveal new Complexity

    NASA Astrophysics Data System (ADS)

    Sinclair, D. J.; Risk, M.

    2004-12-01

    A mixed kinetic/equilibrium steady state model of trace-element co-precipitation in coral skeleton is presented, and tested against high spatial resolution observations of coral trace-element composition made previously by LA-ICP-MS. The model is implemented in PHREEQC, and simulates physicochemical precipitation from a small pocket of seawater which is isolated by the coral, and modified by enzyme exchange of 2 H+ for Ca2+. The model assumes that all aqueous trace-element species in the calcifying fluid are in full equilibrium and that selected trace element species compete kinetically for precipitation with the major aqueous species (Ca2+ for cation substituents and CO32- for anion substituents). No equilibrium is assumed for the CaCO3 skeleton. Carbon is supplied to the system by diffusion of CO2 into the high-pH calcifying fluid, and trace elements are continuously replenished through the addition of fresh seawater. The rate at which the coral operates the enzyme pump, and the rate at which it replenishes the seawater component are independent variables, and the steady state trace-element composition of the skeleton/calcifying fluid are evaluated over a 2D grid of variables spanning realistic rates of pumping and seawater influx. It is assumed that variations in the trace element composition of the coral skeleton are the result of shifts in the steady-state caused by changes to these variables. The results indicate an unexpected complexity in the response of the trace elements. First order predictions suggest that increasing the rate of calcification by increasing the enzyme pumping should result in a mutual dilution of most trace element species by pumped Ca2+ and diffused CO2. However, for high rates of pumping and low seawater replenishment, the model predicts a change in the trace-element response of the system as high CO32- concentrations drive calcification and deplete Ca2+. This added complexity makes rationalizing observations with models more difficult.

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

    NASA Astrophysics Data System (ADS)

    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.

  5. Unexpected Spin-Crossover and a Low-Pressure Phase Change in an Iron(II)/Dipyrazolylpyridine Complex Exhibiting a High-Spin Jahn- Teller Distortion.

    PubMed

    Kershaw Cook, Laurence J; Thorp-Greenwood, Flora L; Comyn, Tim P; Cespedes, Oscar; Chastanet, Guillaume; Halcrow, Malcolm A

    2015-07-01

    The synthesis of 4-methyl-2,6-di(pyrazol-1-yl)pyridine (L) and four salts of [FeL2]X2 (X– = BF(4)(–), 1; X– = ClO(4)(–), 2; X– = PF(6)(–), 3; X– = CF3SO(3)(–), 4) are reported. Powder samples of 1 and 2 both exhibit abrupt, hysteretic spin-state transitions on cooling, with T(1/2)↓ = 204 and T(1/2)↑ = 209 K (1), and T(1/2)↓ = 175 and T(1/2)↑ = 193 K (2). The 18 K thermal hysteresis loop for 2 is unusually wide for a complex of this type. Single crystal structures of 2 show it to exhibit a Jahn–Teller-distorted six-coordinate geometry in its high-spin state, which would normally inhibit spin-crossover. Bulk samples of 1 and 2 are isostructural by X-ray powder diffraction, and undergo a crystallographic phase change during their spin-transitions. At temperatures below T(1/2), exposing both compounds to 10(–5) Torr pressure inside the powder diffractometer causes a reversible transformation back to the high-temperature crystal phase. Consideration of thermodynamic data implies this cannot be accompanied by a low → high spin-state change, however. Both compounds also exhibit the LIESST effect, with 2 exhibiting an unusually high T(LIESST) of 112 K. The salts 3 and 4 are respectively high-spin and low-spin between 3 and 300 K, with crystalline 3 exhibiting a more pronounced version of the same Jahn–Teller distortion. PMID:26351707

  6. Unexpected Spin-Crossover and a Low-Pressure Phase Change in an Iron(II)/Dipyrazolylpyridine Complex Exhibiting a High-Spin Jahn-Teller Distortion.

    PubMed

    Kershaw Cook, Laurence J; Thorp-Greenwood, Flora L; Comyn, Tim P; Cespedes, Oscar; Chastanet, Guillaume; Halcrow, Malcolm A

    2015-07-01

    The synthesis of 4-methyl-2,6-di(pyrazol-1-yl)pyridine (L) and four salts of [FeL2]X2 (X(-) = BF4(-), 1; X(-) = ClO4(-), 2; X(-) = PF6(-), 3; X(-) = CF3SO3(-), 4) are reported. Powder samples of 1 and 2 both exhibit abrupt, hysteretic spin-state transitions on cooling, with T1/2↓ = 204 and T1/2↑ = 209 K (1), and T1/2↓ = 175 and T1/2↑ = 193 K (2). The 18 K thermal hysteresis loop for 2 is unusually wide for a complex of this type. Single crystal structures of 2 show it to exhibit a Jahn-Teller-distorted six-coordinate geometry in its high-spin state, which would normally inhibit spin-crossover. Bulk samples of 1 and 2 are isostructural by X-ray powder diffraction, and undergo a crystallographic phase change during their spin-transitions. At temperatures below T1/2, exposing both compounds to 10(-5) Torr pressure inside the powder diffractometer causes a reversible transformation back to the high-temperature crystal phase. Consideration of thermodynamic data implies this cannot be accompanied by a low → high spin-state change, however. Both compounds also exhibit the LIESST effect, with 2 exhibiting an unusually high T(LIESST) of 112 K. The salts 3 and 4 are respectively high-spin and low-spin between 3 and 300 K, with crystalline 3 exhibiting a more pronounced version of the same Jahn-Teller distortion. PMID:26052980

  7. An unexpected tetanus case.

    PubMed

    Ergonul, Onder; Egeli, Demet; Kahyaoglu, Bulent; Bahar, Mois; Etienne, Mill; Bleck, Thomas

    2016-06-01

    1 million cases of tetanus are estimated to occur worldwide each year, with more than 200 000 deaths. Tetanus is a life-threatening but preventable disease caused by a toxin produced by Clostridium tetani-a Gram-positive bacillus found in high concentrations in soil and animal excrement. Tetanus is almost completely preventable by active immunisation, but very rarely unexpected cases can occur in individuals who have been previously vaccinated. We report a case of generalised tetanus in a 22-year-old woman that arose despite the protective antitoxin antibody in her serum. The patient received all her vaccinations in the USA; her last vaccination was 6 years ago. The case was unusual because the patient had received all standard vaccinations, had no defined port of entry at disease onset, and had symptoms lasting for 6 months. Tetanus can present with unusual clinical forms; therefore, the diagnosis and management of this rare but difficult disease should be updated. In this Grand Round, we review the clinical features, epidemiology, treatment, and prognosis of C tetani infections. PMID:27301930

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

    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.

  9. Unexpected light behaviour in periodic segmented waveguides

    NASA Astrophysics Data System (ADS)

    Aschiéri, Pierre; Doya, Valérie

    2011-12-01

    In this article, it is shown that multimode periodic segmented waveguides (PSW) are versatile optical systems in which properties of wave chaos can be highlighted. Numerical wave analysis reveals that structures of quantum phase space of PSW are similar to Poincaré sections which display a mixed phase space where stability islands are surrounded by a chaotic sea. Then, unexpected light behavior can occur such as, input gaussian beams do not diverge during the propagation in a highly multimode waveguide.

  10. Predictive ethoinformatics reveals the complex migratory behaviour of a pelagic seabird, the Manx Shearwater

    PubMed Central

    Freeman, Robin; Dean, Ben; Kirk, Holly; Leonard, Kerry; Phillips, Richard A.; Perrins, Chris M.; Guilford, Tim

    2013-01-01

    Understanding the behaviour of animals in the wild is fundamental to conservation efforts. Advances in bio-logging technologies have offered insights into the behaviour of animals during foraging, migration and social interaction. However, broader application of these systems has been limited by device mass, cost and longevity. Here, we use information from multiple logger types to predict individual behaviour in a highly pelagic, migratory seabird, the Manx Shearwater (Puffinus puffinus). Using behavioural states resolved from GPS tracking of foraging during the breeding season, we demonstrate that individual behaviours can be accurately predicted during multi-year migrations from low cost, lightweight, salt-water immersion devices. This reveals a complex pattern of migratory stopovers: some involving high proportions of foraging, and others of rest behaviour. We use this technique to examine three consecutive years of global migrations, revealing the prominence of foraging behaviour during migration and the importance of highly productive waters during migratory stopover. PMID:23635496

  11. Exome sequencing of ion channel genes reveals complex profiles confounding personal risk assessment in epilepsy.

    PubMed

    Klassen, Tara; Davis, Caleb; Goldman, Alica; Burgess, Dan; Chen, Tim; Wheeler, David; McPherson, John; Bourquin, Traci; Lewis, Lora; Villasana, Donna; Morgan, Margaret; Muzny, Donna; Gibbs, Richard; Noebels, Jeffrey

    2011-06-24

    Ion channel mutations are an important cause of rare Mendelian disorders affecting brain, heart, and other tissues. We performed parallel exome sequencing of 237 channel genes in a well-characterized human sample, comparing variant profiles of unaffected individuals to those with the most common neuronal excitability disorder, sporadic idiopathic epilepsy. Rare missense variation in known Mendelian disease genes is prevalent in both groups at similar complexity, revealing that even deleterious ion channel mutations confer uncertain risk to an individual depending on the other variants with which they are combined. Our findings indicate that variant discovery via large scale sequencing efforts is only a first step in illuminating the complex allelic architecture underlying personal disease risk. We propose that in silico modeling of channel variation in realistic cell and network models will be crucial to future strategies assessing mutation profile pathogenicity and drug response in individuals with a broad spectrum of excitability disorders. PMID:21703448

  12. Revealing hidden clonal complexity in Mycobacterium tuberculosis infection by qualitative and quantitative improvement of sampling.

    PubMed

    Pérez-Lago, L; Palacios, J J; Herranz, M; Ruiz Serrano, M J; Bouza, E; García-de-Viedma, D

    2015-02-01

    The analysis of microevolution events, its functional relevance and impact on molecular epidemiology strategies, constitutes one of the most challenging aspects of the study of clonal complexity in infection by Mycobacterium tuberculosis. In this study, we retrospectively evaluated whether two improved sampling schemes could provide access to the clonal complexity that is undetected by the current standards (analysis of one isolate from one sputum). We evaluated in 48 patients the analysis by mycobacterial interspersed repetitive unit-variable number tandem repeat of M. tuberculosis isolates cultured from bronchial aspirate (BAS) or bronchoalveolar lavage (BAL) and, in another 16 cases, the analysis of a higher number of isolates from independent sputum samples. Analysis of the isolates from BAS/BAL specimens revealed clonal complexity in a very high proportion of cases (5/48); in most of these cases, complexity was not detected when the isolates from sputum samples were analysed. Systematic analysis of isolates from multiple sputum samples also improved the detection of clonal complexity. We found coexisting clonal variants in two of 16 cases that would have gone undetected in the analysis of the isolate from a single sputum specimen. Our results suggest that analysis of isolates from BAS/BAL specimens is highly efficient for recording the true clonal composition of M. tuberculosis in the lungs. When these samples are not available, we recommend increasing the number of isolates from independent sputum specimens, because they might not harbour the same pool of bacteria. Our data suggest that the degree of clonal complexity in tuberculosis has been underestimated because of the deficiencies inherent in a simplified procedure. PMID:25658553

  13. COMBINED DELAY AND GRAPH EMBEDDING OF EPILEPTIC DISCHARGES IN EEG REVEALS COMPLEX AND RECURRENT NONLINEAR DYNAMICS

    PubMed Central

    Erem, B.; Hyde, D.E.; Peters, J.M.; Duffy, F.H.; Brooks, D.H.; Warfield, S.K.

    2015-01-01

    The dynamical structure of the brain’s electrical signals contains valuable information about its physiology. Here we combine techniques for nonlinear dynamical analysis and manifold identification to reveal complex and recurrent dynamics in interictal epileptiform discharges (IEDs). Our results suggest that recurrent IEDs exhibit some consistent dynamics, which may only last briefly, and so individual IED dynamics may need to be considered in order to understand their genesis. This could potentially serve to constrain the dynamics of the inverse source localization problem. PMID:26366250

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

    PubMed Central

    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

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

    PubMed

    Kohda, Masakazu; Tokuzawa, Yoshimi; Kishita, Yoshihito; 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

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

    PubMed

    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

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

    PubMed Central

    Møller, Marie S.; Vester-Christensen, Malene B.; Jensen, Johanne M.; Hachem, Maher Abou; Henriksen, Anette; Svensson, Birte

    2015-01-01

    Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, 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 of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs. PMID:25792743

  18. Crystal structure of barley limit dextrinase-limit dextrinase inhibitor (LD-LDI) complex reveals insights into mechanism and diversity of cereal type inhibitors.

    PubMed

    Møller, Marie S; Vester-Christensen, Malene B; Jensen, Johanne M; Hachem, Maher Abou; Henriksen, Anette; Svensson, Birte

    2015-05-15

    Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, 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 of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs. PMID:25792743

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

    SciTech Connect

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

    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.

  20. Structure of a topoisomerase II-DNA-nucleotide complex reveals a new control mechanism for ATPase activity.

    PubMed

    Schmidt, Bryan H; Osheroff, Neil; Berger, James M

    2012-11-01

    Type IIA topoisomerases control DNA supercoiling and disentangle chromosomes through a complex ATP-dependent strand-passage mechanism. Although a general framework exists for type IIA topoisomerase function, the architecture of the full-length enzyme has remained undefined. Here we present the structure of a fully catalytic Saccharomyces cerevisiae topoisomerase II homodimer complexed with DNA and a nonhydrolyzable ATP analog. The enzyme adopts a domain-swapped configuration wherein the ATPase domain of one protomer sits atop the nucleolytic region of its partner subunit. This organization produces an unexpected interaction between bound DNA and a conformational transducing element in the ATPase domain, which we show is critical for both DNA-stimulated ATP hydrolysis and global topoisomerase activity. Our data indicate that the ATPase domains pivot about each other to ensure unidirectional strand passage and that this state senses bound DNA to promote ATP turnover and enzyme reset. PMID:23022727

  1. Are Rogue Waves Really Unexpected?

    NASA Astrophysics Data System (ADS)

    Fedele, Francesco

    2016-05-01

    An unexpected wave is defined by Gemmrich & Garrett (2008) as a wave that is much taller than a set of neighboring waves. Their definition of "unexpected" refers to a wave that is not anticipated by a casual observer. Clearly, unexpected waves defined in this way are predictable in a statistical sense. They can occur relatively often with a small or moderate crest height, but large unexpected waves that are rogue are rare. Here, this concept is elaborated and statistically described based on a third-order nonlinear model. In particular, the conditional return period of an unexpected wave whose crest exceeds a given threshold is developed. This definition leads to greater return periods or on average less frequent occurrences of unexpected waves than those implied by the conventional return periods not conditioned on a reference threshold. Ultimately, it appears that a rogue wave that is also unexpected would have a lower occurrence frequency than that of a usual rogue wave. As specific applications, the Andrea and WACSIS rogue wave events are examined in detail. Both waves appeared without warning and their crests were nearly $2$-times larger than the surrounding $O(10)$ wave crests, and thus unexpected. The two crest heights are nearly the same as the threshold~$h_{0.3\\cdot10^{6}}\\sim1.6H_{s}$ exceeded on average once every~$0.3\\cdot 10^{6}$ waves, where $H_s$ is the significant wave height. In contrast, the Andrea and WACSIS events, as both rogue and unexpected, would occur slightly less often and on average once every~$3\\cdot10^{6}$ and~$0.6\\cdot10^6$ waves respectively.

  2. Comparative Venomics Reveals the Complex Prey Capture Strategy of the Piscivorous Cone Snail Conus catus.

    PubMed

    Himaya, S W A; Jin, Ai-Hua; Dutertre, Sébastien; Giacomotto, Jean; Mohialdeen, Hoshyar; Vetter, Irina; Alewood, Paul F; Lewis, Richard J

    2015-10-01

    Venomous marine cone snails produce a unique and remarkably diverse range of venom peptides (conotoxins and conopeptides) that have proven to be invaluable as pharmacological probes and leads to new therapies. Conus catus is a hook-and-line fish hunter from clade I, with ∼20 conotoxins identified, including the analgesic ω-conotoxin CVID (AM336). The current study unravels the venom composition of C. catus with tandem mass spectrometry and 454 sequencing data. From the venom gland transcriptome, 104 precursors were recovered from 11 superfamilies, with superfamily A (especially κA-) conotoxins dominating (77%) their venom. Proteomic analysis confirmed that κA-conotoxins dominated the predation-evoked milked venom of each of six C. catus analyzed and revealed remarkable intraspecific variation in both the intensity and type of conotoxins. High-throughput FLIPR assays revealed that the predation-evoked venom contained a range of conotoxins targeting the nAChR, Cav, and Nav ion channels, consistent with α- and ω-conotoxins being used for predation by C. catus. However, the κA-conotoxins did not act at these targets but induced potent and rapid immobilization followed by bursts of activity and finally paralysis when injected intramuscularly in zebrafish. Our venomics approach revealed the complexity of the envenomation strategy used by C. catus, which contains a mix of both excitatory and inhibitory venom peptides. PMID:26322961

  3. Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ

    PubMed Central

    Pertsinidis, Alexandros; Mukherjee, Konark; Sharma, Manu; Pang, Zhiping P.; Park, Sang Ryul; Zhang, Yunxiang; Brunger, Axel T.; Südhof, Thomas C.; Chu, Steven

    2013-01-01

    Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ. PMID:23821748

  4. Quantitative proteomics reveal ATM kinase-dependent exchange in DNA damage response complexes.

    PubMed

    Choi, Serah; Srivas, Rohith; Fu, Katherine Y; Hood, Brian L; Dost, Banu; Gibson, Gregory A; Watkins, Simon C; Van Houten, Bennett; Bandeira, Nuno; Conrads, Thomas P; Ideker, Trey; Bakkenist, Christopher J

    2012-10-01

    ATM is a protein kinase that initiates a well-characterized signaling cascade in cells exposed to ionizing radiation (IR). However, the role for ATM in coordinating critical protein interactions and subsequent exchanges within DNA damage response (DDR) complexes is unknown. We combined SILAC-based tandem mass spectrometry and a subcellular fractionation protocol to interrogate the proteome of irradiated cells treated with or without the ATM kinase inhibitor KU55933. We developed an integrative network analysis to identify and prioritize proteins that were responsive to KU55933, specifically in chromatin, and that were also enriched for physical interactions with known DNA repair proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as strong candidates. Using fluorescence recovery after photobleaching, we found that the exchange of GFP-53BP1 in DDR complexes decreased with KU55933. Further, we found that ANXA1 knockdown sensitized cells to IR via a mechanism that was not potentiated by KU55933. Our study reveals a role for ATM kinase activity in the dynamic exchange of proteins in DDR complexes and identifies a role for ANXA1 in cellular radioprotection. PMID:22909323

  5. Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening

    PubMed Central

    Sadre-Bazzaz, Kianoush; Whitby, Frank G.; Robinson, Howard; Formosa, Tim; Hill, Christopher P.

    2010-01-01

    Summary 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 3.4Å 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, challenges 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. PMID:20227375

  6. Structural analysis of the RZZ complex reveals common ancestry with multisubunit vesicle tethering machinery.

    PubMed

    Civril, Filiz; Wehenkel, Annemarie; Giorgi, Federico M; Santaguida, Stefano; Di Fonzo, Andrea; Grigorean, Gabriela; Ciccarelli, Francesca D; Musacchio, Andrea

    2010-05-12

    The RZZ complex recruits dynein to kinetochores. We investigated structure, topology, and interactions of the RZZ subunits (ROD, ZWILCH, and ZW10) in vitro, in vivo, and in silico. We identify neuroblastoma-amplified gene (NAG), a ZW10 binder, as a ROD homolog. ROD and NAG contain an N-terminal beta propeller followed by an alpha solenoid, which is the architecture of certain nucleoporins and vesicle coat subunits, suggesting a distant evolutionary relationship. ZW10 binding to ROD and NAG is mutually exclusive. The resulting ZW10 complexes (RZZ and NRZ) respectively contain ZWILCH and RINT1 as additional subunits. The X-ray structure of ZWILCH, the first for an RZZ subunit, reveals a novel fold distinct from RINT1's. The evolutionarily conserved NRZ likely acts as a tethering complex for retrograde trafficking of COPI vesicles from the Golgi to the endoplasmic reticulum. The RZZ, limited to metazoans, probably evolved from the NRZ, exploiting the dynein-binding capacity of ZW10 to direct dynein to kinetochores. PMID:20462495

  7. A spider species complex revealed high cryptic diversity in South China caves.

    PubMed

    Zhang, Yuanyuan; Li, Shuqiang

    2014-10-01

    Cryptic species, which are an important component of biodiversity, have rarely been studied in South China karst. We investigated cryptic diversity in the cave species complex Telema cucurbitina, which has a narrow niche but widespread distribution among multiple caves. We sampled another 15 populations (caves) in addition to the population from the type locality. Phylogenetic results indicated that individuals from the same cave constituted well-supported clades. Species diversity within this species complex was assessed in a coalescent framework, first with a Bayesian extension of the general mixed Yule coalescent (bGMYC) model and a Bayesian species delimitation method (BPP). Both species delimitation methods identified each cave population as a separate species. We propose that each cave population within this species complex was a separate evolving lineage and therefore 16 OTUs were recovered based on our molecular data despite their high morphological similarities. We also propose that the unrecognized organism's diversity within South China caves might be extremely large considering our case. Furthermore, our work reveals that species discovery of cave organisms by morphological data has a high probability of underestimating hidden diversity. Our work also highlights the need for conservation strategies to protect this largely neglected diversity of cave organisms. PMID:24994029

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

    NASA Astrophysics Data System (ADS)

    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

  9. Novel binding motif and new flexibility revealed by structural analyses of a pyruvate dehydrogenase-dihydrolipoyl acetyltransferase subcomplex from the Escherichia coli pyruvate dehydrogenase multienzyme complex.

    PubMed

    Arjunan, Palaniappa; Wang, Junjie; Nemeria, Natalia S; Reynolds, Shelley; Brown, Ian; Chandrasekhar, Krishnamoorthy; Calero, Guillermo; Jordan, Frank; Furey, William

    2014-10-24

    The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly. PMID:25210042

  10. A multi-gene approach reveals a complex evolutionary history in the Cyanistes species group.

    PubMed

    Illera, Juan Carlos; Koivula, Kari; Broggi, Juli; Päckert, Martin; Martens, Jochen; Kvist, Laura

    2011-10-01

    Quaternary climatic oscillations have been considered decisive in shaping much of the phylogeographic structure around the Mediterranean Basin. Within this paradigm, peripheral islands are usually considered as the endpoints of the colonization processes. Here, we use nuclear and mitochondrial markers to investigate the phylogeography of the blue tit complex (blue tit Cyanistes caeruleus, Canary blue tit C. teneriffae and azure tit C. cyanus), and assess the role of the Canary Islands for the geographic structuring of genetic variation. The Canary blue tit exhibits strong genetic differentiation within the Canary Islands and, in combination with other related continental species, provides an ideal model in which to examine recent differentiation within a closely related group of continental and oceanic island avian species. We analysed DNA sequences from 51 breeding populations and more than 400 individuals in the blue tit complex. Discrepancies in the nuclear and mitochondrial gene trees provided evidence of a complex evolutionary process around the Mediterranean Basin. Coalescent analyses revealed gene flow between C. caeruleus and C. teneriffae suggesting a dynamic process with multiple phases of colonization and geographic overlapping ranges. Microsatellite data indicated strong genetic differentiation among the Canary Islands and between the Canary archipelago and the close continental areas, indicating limited contemporary gene flow. Diversification of the blue tit complex is estimated to have started during the early Pliocene (≈ 5 Ma), coincident with the end of Messinian salinity crisis. Phylogenetic analyses indicated that the North African blue tit is derived from the Canary blue tits, a pattern is avian 'back colonization' that contrasts with more traditionally held views of islands being sinks rather than sources. PMID:21880092

  11. Genetic Networking of the Bemisia tabaci Cryptic Species Complex Reveals Pattern of Biological Invasions

    PubMed Central

    De Barro, Paul; Ahmed, Muhammad Z.

    2011-01-01

    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 the available

  12. The complex hybrid origins of the root knot nematodes revealed through comparative genomics

    PubMed Central

    Kumar, Sujai; Koutsovoulos, Georgios; Blaxter, Mark L.

    2014-01-01

    Root knot nematodes (RKN) can infect most of the world’s agricultural crop species and are among the most important of all plant pathogens. As yet however we have little understanding of their origins or the genomic basis of their extreme polyphagy. The most damaging pathogens reproduce by obligatory mitotic parthenogenesis and it has been suggested that these species originated from interspecific hybridizations between unknown parental taxa. We have sequenced the genome of the diploid meiotic parthenogen Meloidogyne floridensis, and use a comparative genomic approach to test the hypothesis that this species was involved in the hybrid origin of the tropical mitotic parthenogen Meloidogyne incognita. Phylogenomic analysis of gene families from M. floridensis, M. incognita and an outgroup species Meloidogyne hapla was carried out to trace the evolutionary history of these species’ genomes, and we demonstrate that M. floridensis was one of the parental species in the hybrid origins of M. incognita. Analysis of the M. floridensis genome itself revealed many gene loci present in divergent copies, as they are in M. incognita, indicating that it too had a hybrid origin. The triploid M. incognita is shown to be a complex double-hybrid between M. floridensis and a third, unidentified, parent. The agriculturally important RKN have very complex origins involving the mixing of several parental genomes by hybridization and their extreme polyphagy and success in agricultural environments may be related to this hybridization, producing transgressive variation on which natural selection can act. It is now clear that studying RKN variation via individual marker loci may fail due to the species’ convoluted origins, and multi-species population genomics is essential to understand the hybrid diversity and adaptive variation of this important species complex. This comparative genomic analysis provides a compelling example of the importance and complexity of hybridization in

  13. Multiple sampling and discriminatory fingerprinting reveals clonally complex and compartmentalized infections by M. bovis in cattle.

    PubMed

    Navarro, Yurena; Romero, Beatriz; Copano, María Francisca; Bouza, Emilio; Domínguez, Lucas; de Juan, Lucía; García-de-Viedma, Darío

    2015-01-30

    The combination of new genotyping tools and a more exhaustive sampling policy in the analysis of infection by Mycobacterium tuberculosis has shown that infection by this pathogen is more complex than initially expected. Mixed infections, coexistence of clonal variants from a parental strain, and compartmentalized infections are all different modalities of this clonal complexity. Until recently, genotyping of Mycobacterium bovis in animal populations was based on spoligotyping and analysis of a single isolate per infection; therefore, clonal complexity is probably underdetected. We used multiple sampling combined with highly discriminatory MIRU-VNTR to study compartmentalized infections by M. bovis in a low-tuberculosis prevalence setting. We spoligotyped the M. bovis isolates from two or more anatomic locations sampled from 55 animals on 39 independent farms. Compartmentalized infections, with two different strains infecting independent lymph nodes in the same animal, were found in six cases (10.9%). MIRU-VNTR analysis confirmed that the compartmentalization was strict and that only one strain was present in each infected node. MIRU-VNTR analysis of additional infected animals on one of the farms confirmed that the compartmentalized infection was a consequence of superinfection, since the two strains were independently infecting other animals. This same analysis revealed the emergence of a microevolved clonal variant in one of the lymph nodes of the compartmentalized animal. Clonal complexity must also be taken into consideration in M. bovis infection, even in low-prevalence settings, and analyses must be adapted to detect it and increase the accuracy of molecular epidemiology studies. PMID:25439651

  14. Unexpected Molecular Weight Effect in Polymer Nanocomposites

    NASA Astrophysics Data System (ADS)

    Cheng, Shiwang; Holt, Adam P.; Wang, Huiqun; Fan, Fei; Bocharova, Vera; Martin, Halie; Etampawala, Thusitha; White, B. Tyler; Saito, Tomonori; Kang, Nam-Goo; Dadmun, Mark D.; Mays, Jimmy W.; Sokolov, Alexei P.

    2016-01-01

    The properties of the interfacial layer between the polymer matrix and nanoparticles largely determine the macroscopic properties of polymer nanocomposites (PNCs). Although the static thickness of the interfacial layer was found to increase with the molecular weight (MW), the influence of MW on segmental relaxation and the glass transition in this layer remains to be explored. In this Letter, we show an unexpected MW dependence of the interfacial properties in PNC with attractive polymer-nanoparticle interactions: the thickness of the interfacial layer with hindered segmental relaxation decreases as MW increases, in sharp contrast to theoretical predictions. Further analyses reveal a reduction in mass density of the interfacial layer with increasing MW, which can elucidate these unexpected dynamic effects. Our observations call for a significant revision of the current understandings of PNCs and suggest interesting ways to tailor their properties.

  15. Unexpected molecular weight effect in polymer nanocomposites

    DOE PAGESBeta

    Cheng, Shiwang; Holt, Adam P.; Wang, Huiqun; Fan, Fei; Bocharova, Vera; Martin, Halie J.; Etampawala, Thusitha N.; White, Benjamin Tyler; Saito, Tomonori; Kang, Nam -Goo; et al

    2016-01-22

    Here, the properties of the interfacial layer between the polymer matrix and nanoparticles largely determine the macroscopic properties of polymer nanocomposites (PNCs). Although the static thickness of the interfacial layer was found to increase with the molecular weight (MW), the influence of MW on segmental relaxation and the glass transition in this layer remains to be explored. In this Letter, we show an unexpected MW dependence of the interfacial properties in PNC with attractive polymer-nanoparticle interactions: the thickness of the interfacial layer with hindered segmental relaxation decreases as MW increases, in sharp constrast to theoretical predictions. Further analyses reveal amore » reduction in mass density of the interfacial layer with increasing MW, which can explain these unexpected dynamic effects. Our observations call for a significant revision of the current understandings of PNCs and suggest interesting ways to tailor their properties.« less

  16. Unexpected Molecular Weight Effect in Polymer Nanocomposites.

    PubMed

    Cheng, Shiwang; Holt, Adam P; Wang, Huiqun; Fan, Fei; Bocharova, Vera; Martin, Halie; Etampawala, Thusitha; White, B Tyler; Saito, Tomonori; Kang, Nam-Goo; Dadmun, Mark D; Mays, Jimmy W; Sokolov, Alexei P

    2016-01-22

    The properties of the interfacial layer between the polymer matrix and nanoparticles largely determine the macroscopic properties of polymer nanocomposites (PNCs). Although the static thickness of the interfacial layer was found to increase with the molecular weight (MW), the influence of MW on segmental relaxation and the glass transition in this layer remains to be explored. In this Letter, we show an unexpected MW dependence of the interfacial properties in PNC with attractive polymer-nanoparticle interactions: the thickness of the interfacial layer with hindered segmental relaxation decreases as MW increases, in sharp contrast to theoretical predictions. Further analyses reveal a reduction in mass density of the interfacial layer with increasing MW, which can elucidate these unexpected dynamic effects. Our observations call for a significant revision of the current understandings of PNCs and suggest interesting ways to tailor their properties. PMID:26849618

  17. Unexpected higher stabilisation of two classical antiaromatic frameworks with a ruthenium fragment compared to the osmium counterpart: origin probed by DFT calculations.

    PubMed

    Wu, Jingjing; Hao, Yulei; An, Ke; Zhu, Jun

    2016-01-01

    Density functional theory (DFT) calculations were carried out to investigate the stability and aromaticity of metallapentalocyclobutadienes. The results reveal unexpected higher stabilisation achieved with a 4d ruthenium fragment compared to the 5d [corrected] osmium counterpart. Moreover, direct 1-3 metal-carbon bonding in the metallabutadiene unit of these two complexes is negligible. PMID:26505956

  18. Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence

    PubMed Central

    2010-01-01

    Background The Ralstonia solanacearum species complex includes thousands of strains pathogenic to an unusually wide range of plant species. These globally dispersed and heterogeneous strains cause bacterial wilt diseases, which have major socio-economic impacts. Pathogenicity is an ancestral trait in R. solanacearum and strains with high genetic variation can be subdivided into four phylotypes, correlating to isolates from Asia (phylotype I), the Americas (phylotype IIA and IIB), Africa (phylotype III) and Indonesia (phylotype IV). Comparison of genome sequences strains representative of this phylogenetic diversity can help determine which traits allow this bacterium to be such a pathogen of so many different plant species and how the bacteria survive in many different habitats. Results The genomes of three tomato bacterial wilt pathogens, CFBP2957 (phy. IIA), CMR15 (phy. III) and PSI07 (phy. IV) were sequenced and manually annotated. These genomes were compared with those of three previously sequenced R. solanacearum strains: GMI1000 (tomato, phy. I), IPO1609 (potato, phy. IIB), and Molk2 (banana, phy. IIB). The major genomic features (size, G+C content, number of genes) were conserved across all of the six sequenced strains. Despite relatively high genetic distances (calculated from average nucleotide identity) and many genomic rearrangements, more than 60% of the genes of the megaplasmid and 70% of those on the chromosome are syntenic. The three new genomic sequences revealed the presence of several previously unknown traits, probably acquired by horizontal transfers, within the genomes of R. solanacearum, including a type IV secretion system, a rhi-type anti-mitotic toxin and two small plasmids. Genes involved in virulence appear to be evolving at a faster rate than the genome as a whole. Conclusions Comparative analysis of genome sequences and gene content confirmed the differentiation of R. solanacearum species complex strains into four phylotypes. Genetic

  19. Unexpected angular or rotational deformity after corrective osteotomy

    PubMed Central

    2014-01-01

    Background Codman’s paradox reveals a misunderstanding of geometry in orthopedic practice. Physicians often encounter situations that cannot be understood intuitively during orthopedic interventions such as corrective osteotomy. Occasionally, unexpected angular or rotational deformity occurs during surgery. This study aimed to draw the attention of orthopedic surgeons toward the concepts of orientation and rotation and demonstrate the potential for unexpected deformity after orthopedic interventions. This study focused on three situations: shoulder arthrodesis, femoral varization derotational osteotomy, and femoral derotation osteotomy. Methods First, a shoulder model was generated to calculate unexpected rotational deformity to demonstrate Codman’s paradox. Second, femoral varization derotational osteotomy was simulated using a cylinder model. Third, a reconstructed femoral model was used to calculate unexpected angular or rotational deformity during femoral derotation osteotomy. Results Unexpected external rotation was found after forward elevation and abduction of the shoulder joint. In the varization and derotation model, closed-wedge osteotomy and additional derotation resulted in an unexpected extension and valgus deformity, namely, under-correction of coxa valga. After femoral derotational osteotomy, varization and extension of the distal fragment occurred, although the extension was negligible. Conclusions Surgeons should be aware of unexpected angular deformity after surgical procedure involving bony areas. The degree of deformity differs depending on the context of the surgical procedure. However, this study reveals that notable deformities can be expected during orthopedic procedures such as femoral varization derotational osteotomy. PMID:24886469

  20. Complex (Nonstandard) Six-Layer Polytypes of Lizardite Revealed from Oblique-Texture Electron Diffraction Patterns

    SciTech Connect

    Zhukhlistov, A.P.; Zinchuk, N.N.; Kotel'nikov, D.D.

    2004-11-01

    Association of simple (1T and 3R) and two complex (nonstandard) orthogonal polytypes of the serpentine mineral lizardite from the Catoca kimberlite pipe (West Africa) association is revealed from oblique-texture electron diffraction patterns. A six-layer polytype with an ordered superposition of equally oriented layers (notation 3{sub 2}3{sub 2}3{sub 4}3{sub 4}3{sub 6}3{sub 6} or ++ - -00) belonging to the structural group A and a three-layer (336 or I,I,II) or a six-layer (336366 or I,I,II,I,II,II) polytype with alternating oppositely oriented layers and semi-disordered structure are identified using polytype analysis.

  1. Synthetic Nucleosomes Reveal that GlcNAcylation Modulates Direct Interaction with the FACT Complex.

    PubMed

    Raj, Ritu; Lercher, Lukas; Mohammed, Shabaz; Davis, Benjamin G

    2016-07-25

    Transcriptional regulation can be established by various post-translational modifications (PTMs) on histone proteins in the nucleosome and by nucleobase modifications on chromosomal DNA. Functional consequences of histone O-GlcNAcylation (O-GlcNAc=O-linked β-N-acetylglucosamine) are largely unexplored. Herein, we generate homogeneously GlcNAcylated histones and nucleosomes by chemical post-translational modification. Mass-spectrometry-based quantitative interaction proteomics reveals a direct interaction between GlcNAcylated nucleosomes and the "facilitates chromatin transcription" (FACT) complex. Preferential binding of FACT to GlcNAcylated nucleosomes may point towards O-GlcNAcylation as one of the triggers for FACT-driven transcriptional control. PMID:27272618

  2. Crystal structure of a transcribing RNA Polymerase II complex reveals a complete transcription bubble

    PubMed Central

    Barnes, Christopher O.; Calero, Monica; Malik, Indranil; Graham, Brian W.; Spahr, Henrik; Lin, Guowu; Cohen, Aina; Brown, Ian S.; Zhang, Qiangmin; Pullara, Filippo; Trakselis, Michael A.; Kaplan, Craig D.; Calero, Guillermo

    2015-01-01

    Summary Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop-1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the Trigger Loop (TL), allowing visualization of its open state. Overall, our observations suggest that “open/closed” conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation. PMID:26186291

  3. Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics

    PubMed Central

    Streets, Aaron M.; Sourigues, Yannick; Kopito, Ron R.; Melki, Ronald; Quake, Stephen R.

    2013-01-01

    An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation. PMID:23349924

  4. The Integrative Taxonomic Approach Reveals Host Specific Species in an Encyrtid Parasitoid Species Complex

    PubMed Central

    Chesters, Douglas; Wang, Ying; Yu, Fang; Bai, Ming; Zhang, Tong-Xin; Hu, Hao-Yuan; Zhu, Chao-Dong; Li, Cheng-De; Zhang, Yan-Zhou

    2012-01-01

    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. PMID:22666375

  5. Comparative mapping in the Poaceae family reveals translocations in the complex polyploid genome of sugarcane

    PubMed Central

    2014-01-01

    Background The understanding of sugarcane genetics has lagged behind that of other members of the Poaceae family such as wheat, rice, barley and sorghum mainly due to the complexity, size and polyploidization of the genome. We have used the genetic map of a sugarcane cultivar to generate a consensus genetic map to increase genome coverage for comparison to the sorghum genome. We have utilized the recently developed sugarcane DArT array to increase the marker density within the genetic map. The sequence of these DArT markers plus SNP and EST-SSR markers was then used to form a bridge to the sorghum genomic sequence by BLAST alignment to start to unravel the complex genomic architecture of sugarcane. Results Comparative mapping revealed that certain sugarcane chromosomes show greater levels of synteny to sorghum than others. On a macrosyntenic level a good collinearity was observed between sugarcane and sorghum for 4 of the 8 homology groups (HGs). These 4 HGs were syntenic to four sorghum chromosomes with from 98% to 100% of these chromosomes covered by these linked markers. Four major chromosome rearrangements were identified between the other four sugarcane HGs and sorghum, two of which were condensations of chromosomes reducing the basic chromosome number of sugarcane from x = 10 to x = 8. This macro level of synteny was transferred to other members within the Poaceae family such as maize to uncover the important evolutionary relationships that exist between sugarcane and these species. Conclusions Comparative mapping of sugarcane to the sorghum genome has revealed new information on the genome structure of sugarcane which will help guide identification of important genes for use in sugarcane breeding. Furthermore of the four major chromosome rearrangements identified in this study, three were common to maize providing some evidence that chromosome reduction from a common paleo-ancestor of both maize and sugarcane was driven by the same translocation

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

    SciTech Connect

    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.

  7. Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq

    PubMed Central

    Shepard, Peter J.; Choi, Eun-A; Lu, Jente; Flanagan, Lisa A.; Hertel, Klemens J.; Shi, Yongsheng

    2011-01-01

    Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%–50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3′ untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation. PMID:21343387

  8. What does population structure analysis reveal about the Pterostylis longifolia complex (Orchidaceae)?

    PubMed Central

    Janes, Jasmine K; Steane, Dorothy A; Vaillancourt, René E

    2012-01-01

    Morphologically similar groups of species are common and pose significant challenges for taxonomists. Differences in approaches to classifying unique species can result in some species being overlooked, whereas others are wrongly conserved. The genetic diversity and population structure of the Pterostylis longifolia complex (Orchidaceae) in Tasmania was investigated to determine if four species, and potential hybrids, could be distinguished through genomic AFLP and chloroplast restriction-fragment-length polymorphism (RFLP) markers. Analysis of molecular variance (AMOVA) results indicated that little genetic variation was present among taxa, whereas PCoA analyses revealed genetic variation at a regional scale irrespective of taxa. Population genetic structure analyses identified three clusters that correspond to regional genetic and single taxon-specific phenotypic variation. The results from this study suggest that “longifolia” species have persisted throughout the last glacial maximum in Tasmania and that the complex may be best treated as a single taxon with several morphotypes. These results could have serious evolutionary and conservation implications as taxonomic changes could result in the instatement of a single, widespread taxon in which rarer morphotypes are not protected. PMID:23170201

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

    PubMed

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

    2016-01-01

    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. PMID:26857477

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

    NASA Astrophysics Data System (ADS)

    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.

  11. Differential Network Analysis Reveals Evolutionary Complexity in Secondary Metabolism of Rauvolfia serpentina over Catharanthus roseus.

    PubMed

    Pathania, Shivalika; Bagler, Ganesh; Ahuja, Paramvir S

    2016-01-01

    Comparative co-expression analysis of multiple species using high-throughput data is an integrative approach to determine the uniformity as well as diversification in biological processes. Rauvolfia serpentina and Catharanthus roseus, both members of Apocyanacae family, are reported to have remedial properties against multiple diseases. Despite of sharing upstream of terpenoid indole alkaloid pathway, there is significant diversity in tissue-specific synthesis and accumulation of specialized metabolites in these plants. This led us to implement comparative co-expression network analysis to investigate the modules and genes responsible for differential tissue-specific expression as well as species-specific synthesis of metabolites. Toward these goals differential network analysis was implemented to identify candidate genes responsible for diversification of metabolites profile. Three genes were identified with significant difference in connectivity leading to differential regulatory behavior between these plants. These genes may be responsible for diversification of secondary metabolism, and thereby for species-specific metabolite synthesis. The network robustness of R. serpentina, determined based on topological properties, was also complemented by comparison of gene-metabolite networks of both plants, and may have evolved to have complex metabolic mechanisms as compared to C. roseus under the influence of various stimuli. This study reveals evolution of complexity in secondary metabolism of R. serpentina, and key genes that contribute toward diversification of specific metabolites. PMID:27588023

  12. Systematic Analysis of Dimeric E3-RING Interactions Reveals Increased Combinatorial Complexity in Human Ubiquitination Networks*

    PubMed Central

    Woodsmith, Jonathan; Jenn, Robert C.; Sanderson, Chris M.

    2012-01-01

    Ubiquitination controls the stability or function of many human proteins, thereby regulating a wide range of physiological processes. In most cases the combinatorial pattern of protein interactions that facilitate substrate recognition or modification remain unclear. Moreover, the efficiency of ubiquitination reactions can be altered by the formation of homo- and heterotypic E3-RING complexes. To establish the prevalence and nature of binary E3-RING/E3-RING interactions systematic yeast two-hybrid screens were performed to test 7269 potential interactions between 124 human E3-RING proteins. These studies identified 228 dimeric interactions between 100 E3-RINGs, of which 205 were novel. Complementary co-immunoprecipitation studies were performed to test predicted network interactions, showing a high correlation (64%) with primary yeast two-hybrid data. This data was integrated with known E3-RING interactions, tissue expression profiles and proteomic ubiquitination datasets to facilitate identification of subnetworks in which E3-RING dimerization events have the potential to alter network structure. These results reveal a widespread yet selective pattern of E3-RING dimerization events, which have the potential to confer further combinatorial complexity within human ubiquitination cascades. PMID:22493164

  13. Differential Network Analysis Reveals Evolutionary Complexity in Secondary Metabolism of Rauvolfia serpentina over Catharanthus roseus

    PubMed Central

    Pathania, Shivalika; Bagler, Ganesh; Ahuja, Paramvir S.

    2016-01-01

    Comparative co-expression analysis of multiple species using high-throughput data is an integrative approach to determine the uniformity as well as diversification in biological processes. Rauvolfia serpentina and Catharanthus roseus, both members of Apocyanacae family, are reported to have remedial properties against multiple diseases. Despite of sharing upstream of terpenoid indole alkaloid pathway, there is significant diversity in tissue-specific synthesis and accumulation of specialized metabolites in these plants. This led us to implement comparative co-expression network analysis to investigate the modules and genes responsible for differential tissue-specific expression as well as species-specific synthesis of metabolites. Toward these goals differential network analysis was implemented to identify candidate genes responsible for diversification of metabolites profile. Three genes were identified with significant difference in connectivity leading to differential regulatory behavior between these plants. These genes may be responsible for diversification of secondary metabolism, and thereby for species-specific metabolite synthesis. The network robustness of R. serpentina, determined based on topological properties, was also complemented by comparison of gene-metabolite networks of both plants, and may have evolved to have complex metabolic mechanisms as compared to C. roseus under the influence of various stimuli. This study reveals evolution of complexity in secondary metabolism of R. serpentina, and key genes that contribute toward diversification of specific metabolites. PMID:27588023

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

    DOE PAGESBeta

    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 themore » 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. In conclusion, 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.« less

  15. Brain responses in humans reveal ideal observer-like sensitivity to complex acoustic patterns.

    PubMed

    Barascud, Nicolas; Pearce, Marcus T; Griffiths, Timothy D; Friston, Karl J; Chait, Maria

    2016-02-01

    We use behavioral methods, magnetoencephalography, and functional MRI to investigate how human listeners discover temporal patterns and statistical regularities in complex sound sequences. Sensitivity to patterns is fundamental to sensory processing, in particular in the auditory system, because most auditory signals only have meaning as successions over time. Previous evidence suggests that the brain is tuned to the statistics of sensory stimulation. However, the process through which this arises has been elusive. We demonstrate that listeners are remarkably sensitive to the emergence of complex patterns within rapidly evolving sound sequences, performing on par with an ideal observer model. Brain responses reveal online processes of evidence accumulation--dynamic changes in tonic activity precisely correlate with the expected precision or predictability of ongoing auditory input--both in terms of deterministic (first-order) structure and the entropy of random sequences. Source analysis demonstrates an interaction between primary auditory cortex, hippocampus, and inferior frontal gyrus in the process of discovering the regularity within the ongoing sound sequence. The results are consistent with precision based predictive coding accounts of perceptual inference and provide compelling neurophysiological evidence of the brain's capacity to encode high-order temporal structure in sensory signals. PMID:26787854

  16. Impaired neurodevelopment by the low complexity domain of CPEB4 reveals a convergent pathway with neurodegeneration

    PubMed Central

    Shin, Jihae; Salameh, Johnny S.; Richter, Joel D.

    2016-01-01

    CPEB4 is an RNA binding protein expressed in neuronal tissues including brain and spinal cord. CPEB4 has two domains: one that is structured for RNA binding and one that is unstructured and low complexity that has no known function. Unstructured low complexity domains (LCDs) in proteins are often found in RNA-binding proteins and have been implicated in motor neuron degenerative diseases such as amyotrophic lateral sclerosis, indicating that these regions mediate normal RNA processing as well as pathological events. While CPEB4 null knockout mice are normal, animals expressing only the CPEB4 LCD are neonatal lethal with impaired mobility that display defects in neuronal development such as reduced motor axon branching and abnormal neuromuscular junction formation. Although full-length CPEB4 is nearly exclusively cytoplasmic, the CPEB4 LCD forms nucleolar aggregates and CPEB4 LCD-expressing animals have altered ribosomal RNA biogenesis, ribosomal protein gene expression, and elevated levels of stress response genes such as the actin-bundling protein DRR1, which impedes neurite outgrowth. Some of these features share similarities with other LCD-related neurodegenerative disease. Most strikingly, DRR1 appears to be a common focus of several neurodevelopmental and neurodegenerative disorders. Our study reveals a possible molecular convergence between a neurodevelopmental defect and neurodegeneration mediated by LCDs. PMID:27381259

  17. Impaired neurodevelopment by the low complexity domain of CPEB4 reveals a convergent pathway with neurodegeneration.

    PubMed

    Shin, Jihae; Salameh, Johnny S; Richter, Joel D

    2016-01-01

    CPEB4 is an RNA binding protein expressed in neuronal tissues including brain and spinal cord. CPEB4 has two domains: one that is structured for RNA binding and one that is unstructured and low complexity that has no known function. Unstructured low complexity domains (LCDs) in proteins are often found in RNA-binding proteins and have been implicated in motor neuron degenerative diseases such as amyotrophic lateral sclerosis, indicating that these regions mediate normal RNA processing as well as pathological events. While CPEB4 null knockout mice are normal, animals expressing only the CPEB4 LCD are neonatal lethal with impaired mobility that display defects in neuronal development such as reduced motor axon branching and abnormal neuromuscular junction formation. Although full-length CPEB4 is nearly exclusively cytoplasmic, the CPEB4 LCD forms nucleolar aggregates and CPEB4 LCD-expressing animals have altered ribosomal RNA biogenesis, ribosomal protein gene expression, and elevated levels of stress response genes such as the actin-bundling protein DRR1, which impedes neurite outgrowth. Some of these features share similarities with other LCD-related neurodegenerative disease. Most strikingly, DRR1 appears to be a common focus of several neurodevelopmental and neurodegenerative disorders. Our study reveals a possible molecular convergence between a neurodevelopmental defect and neurodegeneration mediated by LCDs. PMID:27381259

  18. Brain responses in humans reveal ideal observer-like sensitivity to complex acoustic patterns

    PubMed Central

    Pearce, Marcus T.; Griffiths, Timothy D.; Chait, Maria

    2016-01-01

    We use behavioral methods, magnetoencephalography, and functional MRI to investigate how human listeners discover temporal patterns and statistical regularities in complex sound sequences. Sensitivity to patterns is fundamental to sensory processing, in particular in the auditory system, because most auditory signals only have meaning as successions over time. Previous evidence suggests that the brain is tuned to the statistics of sensory stimulation. However, the process through which this arises has been elusive. We demonstrate that listeners are remarkably sensitive to the emergence of complex patterns within rapidly evolving sound sequences, performing on par with an ideal observer model. Brain responses reveal online processes of evidence accumulation—dynamic changes in tonic activity precisely correlate with the expected precision or predictability of ongoing auditory input—both in terms of deterministic (first-order) structure and the entropy of random sequences. Source analysis demonstrates an interaction between primary auditory cortex, hippocampus, and inferior frontal gyrus in the process of discovering the regularity within the ongoing sound sequence. The results are consistent with precision based predictive coding accounts of perceptual inference and provide compelling neurophysiological evidence of the brain's capacity to encode high-order temporal structure in sensory signals. PMID:26787854

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

    PubMed Central

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

    2016-01-01

    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. PMID:26857477

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

    PubMed

    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. PMID:25825752

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

    PubMed Central

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

    2015-01-01

    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. PMID:25825752

  2. Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly

    PubMed Central

    Herrmannová, Anna; Daujotytė, Dalia; Yang, Ji-Chun; Cuchalová, Lucie; Gorrec, Fabrice; Wagner, Susan; Dányi, István; Lukavsky, Peter J.; Shivaya Valášek, Leoš

    2012-01-01

    Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1G107R but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs. PMID:22090426

  3. A detailed analysis of the leaf rolling mutant sll2 reveals complex nature in regulation of bulliform cell development in rice (Oryza sativa L.).

    PubMed

    Zhang, J-J; Wu, S-Y; Jiang, L; Wang, J-L; Zhang, X; Guo, X-P; Wu, C-Y; Wan, J-M

    2015-03-01

    Bulliform cells are large, thin-walled and highly vacuolated cells, and play an important role in controlling leaf rolling in response to drought and high temperature. However, the molecular mechanisms regulating bulliform cell development have not been well documented. Here, we report isolation and characterisation of a rice leaf-rolling mutant, named shallot-like 2 (sll2). The sll2 plants exhibit adaxially rolled leaves, starting from the sixth leaf stage, accompanied by increased photosynthesis and reduced plant height and tiller number. Histological analyses showed shrinkage of bulliform cells, resulting in inward-curved leaves. The mutant is recessive and revertible at a rate of 9%. The leaf rolling is caused by a T-DNA insertion. Cloning of the insertion using TAIL-PCR revealed that the T-DNA was inserted in the promoter region of LOC_Os07 g38664. Unexpectedly, the enhanced expression of LOC_Os07 g38664 by the 35S enhancer in the T-DNA is not responsible for the leaf rolling phenotype. Further, the enhancer also exerted a long-distance effect, including up-regulation of several bulliform cell-related genes. sll2 suppressed the outward leaf rolling of oul1 in the sll2oul1 double mutant. We conclude that leaf rolling in sll2 could be a result of the combined effect of multi-genes, implying a complex network in regulation of bulliform cell development. PMID:25213398

  4. Cryo-EM structure of a tetrameric cyanobacterial photosystem I complex reveals novel subunit interactions.

    PubMed

    Semchonok, Dmitry A; Li, Meng; Bruce, Barry D; Oostergetel, Gert T; Boekema, Egbert J

    2016-09-01

    Photosystem I (PSI) of the thermophilic cyanobacterium Chroococcidiopsis sp. TS-821 (TS-821) forms tetramers Li et al. (2014). Two-dimensional maps obtained by single particle electron microscopy (EM) clearly show that the tetramer lacks four-fold symmetry and is actually composed of a dimer of dimers with C2 symmetry. The resolution of these negative stain 2D maps did not permit the placement of most of the small PSI subunits, except for PsaL. Therefore cryo-EM was used for 3D reconstruction of the PSI tetramer complex. A 3D model at ~11.5Å resolution was obtained and a 2D map within the membrane plane of ~6.1Å. This data was used to build a model that was compared with the high-resolution structure of the PSI of Thermosynechococcus elongatus (T. elongatus) at 2.5Å. This comparison reveals key differences in which subunits are involved in the two different interfaces, interface type 1 within a dimer and interface type 2 between dimers. The type 1 interface in TS-821 is similar to the monomer interface in the trimeric PSI from T. elongatus, with interactions between subunits PsaA, -B, -I, -L and M. In type 2 the interaction is only between PsaA, -B and -L. Unlike the trimeric PSI, the central cavity of the complex is not filled with the PsaL-derived helical bundle, but instead seems filled with lipids. The physiological or evolutionary advantage of the tetramer is unknown. However, the presence of both dimers and tetramers in the thylakoid membrane suggest a dynamic equilibrium that shifts towards the tetramers in high light. PMID:27392600

  5. Tantalizing Thanatos: unexpected links in death pathways.

    PubMed

    Cohen, Isabelle; Castedo, Maria; Kroemer, Guido

    2002-07-01

    Cell death is most frequently the result of apoptosis, an event that is often controlled by mitochondrial membrane permeabilization (MMP). Recent data reveal unexpected functional links between apoptosis and autophagic cell death, in the sense that MMP can trigger autophagy of damaged mitochondria. Conversely, one of the major signal-transducing molecules involved in the activation of autophagy during apoptosis--the so-called DAP kinase--can induce cell death through MMP. Connections are also emerging between apoptosis, autophagy, replicative senescence and cancer-specific metabolic changes. PMID:12185842

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

    NASA Astrophysics Data System (ADS)

    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

  7. Crystal Structure of Clostridium botulinum Whole Hemagglutinin Reveals a Huge Triskelion-shaped Molecular Complex*

    PubMed Central

    Amatsu, Sho; Sugawara, Yo; Matsumura, Takuhiro; Kitadokoro, Kengo; Fujinaga, Yukako

    2013-01-01

    Clostridium botulinum HA is a component of the large botulinum neurotoxin complex and is critical for its oral toxicity. HA plays multiple roles in toxin penetration in the gastrointestinal tract, including protection from the digestive environment, binding to the intestinal mucosal surface, and disruption of the epithelial barrier. At least two properties of HA contribute to these roles: the sugar-binding activity and the barrier-disrupting activity that depends on E-cadherin binding of HA. HA consists of three different proteins, HA1, HA2, and HA3, whose structures have been partially solved and are made up mainly of β-strands. Here, we demonstrate structural and functional reconstitution of whole HA and present the complete structure of HA of serotype B determined by x-ray crystallography at 3.5 Å resolution. This structure reveals whole HA to be a huge triskelion-shaped molecule. Our results suggest that whole HA is functionally and structurally separable into two parts: HA1, involved in recognition of cell-surface carbohydrates, and HA2-HA3, involved in paracellular barrier disruption by E-cadherin binding. PMID:24165130

  8. Complex Contact-Based Dynamics of Microsphere Monolayers Revealed by Resonant Attenuation of Surface Acoustic Waves.

    PubMed

    Hiraiwa, M; Abi Ghanem, M; Wallen, S P; Khanolkar, A; Maznev, A A; Boechler, N

    2016-05-13

    Contact-based vibrations play an essential role in the dynamics of granular materials. Significant insights into vibrational granular dynamics have previously been obtained with reduced-dimensional systems containing macroscale particles. We study contact-based vibrations of a two-dimensional monolayer of micron-sized spheres on a solid substrate that forms a microscale granular crystal. Measurements of the resonant attenuation of laser-generated surface acoustic waves reveal three collective vibrational modes that involve displacements and rotations of the microspheres, as well as interparticle and particle-substrate interactions. To identify the modes, we tune the interparticle stiffness, which shifts the frequency of the horizontal-rotational resonances while leaving the vertical resonance unaffected. From the measured contact resonance frequencies we determine both particle-substrate and interparticle contact stiffnesses and find that the former is an order of magnitude larger than the latter. This study paves the way for investigating complex contact-based dynamics of microscale granular crystals and yields a new approach to studying micro- to nanoscale contact mechanics in multiparticle networks. PMID:27232047

  9. Meditation effects within the hippocampal complex revealed by voxel-based morphometry and cytoarchitectonic probabilistic mapping.

    PubMed

    Luders, Eileen; Kurth, Florian; Toga, Arthur W; Narr, Katherine L; Gaser, Christian

    2013-01-01

    Scientific studies addressing anatomical variations in meditators' brains have emerged rapidly over the last few years, where significant links are most frequently reported with respect to gray matter (GM). To advance prior work, this study examined GM characteristics in a large sample of 100 subjects (50 meditators, 50 controls), where meditators have been practicing close to 20 years, on average. A standard, whole-brain voxel-based morphometry approach was applied and revealed significant meditation effects in the vicinity of the hippocampus, showing more GM in meditators than in controls as well as positive correlations with the number of years practiced. However, the hippocampal complex is regionally segregated by architecture, connectivity, and functional relevance. Thus, to establish differential effects within the hippocampal formation (cornu ammonis, fascia dentata, entorhinal cortex, subiculum) as well as the hippocampal-amygdaloid transition area, we utilized refined cytoarchitectonic probabilistic maps of (peri-) hippocampal subsections. Significant meditation effects were observed within the subiculum specifically. Since the subiculum is known to play a key role in stress regulation and meditation is an established form of stress reduction, these GM findings may reflect neuronal preservation in long-term meditators-perhaps due to an attenuated release of stress hormones and decreased neurotoxicity. PMID:23847572

  10. Complex RNA Folding Kinetics Revealed by Single-Molecule FRET and Hidden Markov Models

    PubMed Central

    2014-01-01

    We have developed a hidden Markov model and optimization procedure for photon-based single-molecule FRET data, which takes into account the trace-dependent background intensities. This analysis technique reveals an unprecedented amount of detail in the folding kinetics of the Diels–Alderase ribozyme. We find a multitude of extended (low-FRET) and compact (high-FRET) states. Five states were consistently and independently identified in two FRET constructs and at three Mg2+ concentrations. Structures generally tend to become more compact upon addition of Mg2+. Some compact structures are observed to significantly depend on Mg2+ concentration, suggesting a tertiary fold stabilized by Mg2+ ions. One compact structure was observed to be Mg2+-independent, consistent with stabilization by tertiary Watson–Crick base pairing found in the folded Diels–Alderase structure. A hierarchy of time scales was discovered, including dynamics of 10 ms or faster, likely due to tertiary structure fluctuations, and slow dynamics on the seconds time scale, presumably associated with significant changes in secondary structure. The folding pathways proceed through a series of intermediate secondary structures. There exist both compact pathways and more complex ones, which display tertiary unfolding, then secondary refolding, and, subsequently, again tertiary refolding. PMID:24568646

  11. Mammalian Axoneme Central Pair Complex Proteins: Broader Roles Revealed by Gene Knockout Phenotypes

    PubMed Central

    Teves, Maria E.; Nagarkatti-Gude, David R.; Zhang, Zhibing; Strauss, Jerome F.

    2016-01-01

    The axoneme genes, their encoded proteins, their functions and the structures they form are largely conserved across species. Much of our knowledge of the function and structure of axoneme proteins in cilia and flagella is derived from studies on model organisms like the green algae, Chlamydomonas reinhardtii. The core structure of cilia and flagella is the axoneme, which in most motile cilia and flagella contains a 9 + 2 configuration of microtubules. The two central microtubules are the scaffold of the central pair complex (CPC). Mutations that disrupt CPC genes in Chlamydomonas and other model organisms result in defects in assembly, stability and function of the axoneme, leading to flagellar motility defects. However, targeted mutations generated in mice in the orthologous CPC genes have revealed significant differences in phenotypes of mutants compared to Chlamydomonas. Here we review observations that support the concept of cell-type specific roles for the CPC genes in mice, and an expanded repertoire of functions for the products of these genes in cilia, including non-motile cilia, and other microtubule-associated cellular functions. PMID:26785425

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

    PubMed

    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. PMID:25615435

  13. Complex Contact-Based Dynamics of Microsphere Monolayers Revealed by Resonant Attenuation of Surface Acoustic Waves

    NASA Astrophysics Data System (ADS)

    Hiraiwa, M.; Abi Ghanem, M.; Wallen, S. P.; Khanolkar, A.; Maznev, A. A.; Boechler, N.

    2016-05-01

    Contact-based vibrations play an essential role in the dynamics of granular materials. Significant insights into vibrational granular dynamics have previously been obtained with reduced-dimensional systems containing macroscale particles. We study contact-based vibrations of a two-dimensional monolayer of micron-sized spheres on a solid substrate that forms a microscale granular crystal. Measurements of the resonant attenuation of laser-generated surface acoustic waves reveal three collective vibrational modes that involve displacements and rotations of the microspheres, as well as interparticle and particle-substrate interactions. To identify the modes, we tune the interparticle stiffness, which shifts the frequency of the horizontal-rotational resonances while leaving the vertical resonance unaffected. From the measured contact resonance frequencies we determine both particle-substrate and interparticle contact stiffnesses and find that the former is an order of magnitude larger than the latter. This study paves the way for investigating complex contact-based dynamics of microscale granular crystals and yields a new approach to studying micro- to nanoscale contact mechanics in multiparticle networks.

  14. Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID

    PubMed Central

    Chen, Allan L.; Kim, Elliot W.; Toh, Justin Y.; Vashisht, Ajay A.; Rashoff, Andrew Q.; Van, Christina; Huang, Amy S.; Moon, Andy S.; Bell, Hannah N.; Bentolila, Laurent A.; Wohlschlegel, James A.

    2015-01-01

    ABSTRACT The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii. PMID:25691595

  15. Dynamic localization of electronic excitation in photosynthetic complexes revealed with chiral two-dimensional spectroscopy

    NASA Astrophysics Data System (ADS)

    Fidler, Andrew F.; Singh, Ved P.; Long, Phillip D.; Dahlberg, Peter D.; Engel, Gregory S.

    2014-02-01

    Time-resolved ultrafast optical probes of chiral dynamics provide a new window allowing us to explore how interactions with such structured environments drive electronic dynamics. Incorporating optical activity into time-resolved spectroscopies has proven challenging because of the small signal and large achiral background. Here we demonstrate that two-dimensional electronic spectroscopy can be adapted to detect chiral signals and that these signals reveal how excitations delocalize and contract following excitation. We dynamically probe the evolution of chiral electronic structure in the light-harvesting complex 2 of purple bacteria following photoexcitation by creating a chiral two-dimensional mapping. The dynamics of the chiral two-dimensional signal directly reports on changes in the degree of delocalization of the excitonic states following photoexcitation. The mechanism of energy transfer in this system may enhance transfer probability because of the coherent coupling among chromophores while suppressing fluorescence that arises from populating delocalized states. This generally applicable spectroscopy will provide an incisive tool to probe ultrafast transient molecular fluctuations that are obscured in non-chiral experiments.

  16. Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes

    PubMed Central

    Labokha, Aksana A.; Osmanović, Dino; Liashkovich, Ivan; Orlova, Elena V.; Ford, Ian J.; Charras, Guillaume; Fassati, Ariberto; Hoogenboom, Bart W.

    2014-01-01

    The nuclear pore complex (NPC) is the gate for transport between the cell nucleus and the cytoplasm. Small molecules cross the NPC by passive diffusion, but molecules larger than ~5 nm must bind to nuclear transport receptors to overcome a selective barrier within the NPC1. Whilst the structure and shape of the cytoplasmic ring of the NPC are relatively well characterized2-5, the selective barrier is situated deep within the central channel of the NPC and depends critically on unstructured nuclear pore proteins5,6, and is therefore not well understood. Here, we show that stiffness topography7 with sharp atomic force microscopy tips can generate nanoscale cross sections of the NPC. The cross sections reveal two distinct structures, a cytoplasmic ring and a central plug structure, which are consistent with the three-dimensional NPC structure derived from electron microscopy2-5. The central plug persists after reactivation of the transport cycle and resultant cargo release, indicating that the plug is an intrinsic part of the NPC barrier. Added nuclear transport receptors accumulate on the intact transport barrier and lead to a homogenization of the barrier stiffness. The observed nanomechanical properties in the NPC indicate the presence of a cohesive barrier to transport, and are quantitatively consistent with the presence of a central condensate of nuclear pore proteins in the NPC channel. PMID:25420031

  17. Simulations reveal that the HIV-1 gp120-CD4 complex dissociates via complex pathways and is a potential target of the polyamidoamine (PAMAM) dendrimer

    NASA Astrophysics Data System (ADS)

    Nandy, Bidisha; Bindu, D. Hima; Dixit, Narendra M.; Maiti, Prabal K.

    2013-07-01

    The polyamidoamine (PAMAM) dendrimer prevents HIV-1 entry into target cells in vitro. Its mechanism of action, however, remains unclear and precludes the design of potent dendrimers targeting HIV-1 entry. We employed steered molecular dynamics simulations to examine whether the HIV-1 gp120-CD4 complex is a target of PAMAM. Our simulations mimicked single molecule force spectroscopy studies of the unbinding of the gp120-CD4 complex under the influence of a controlled external force. We found that the complex dissociates via complex pathways and defies the standard classification of adhesion molecules as catch and slip bonds. When the force loading rate was large, the complex behaved as a slip bond, weakening gradually. When the loading rate was small, the complex initially strengthened, akin to a catch bond, but eventually dissociated over shorter separations than with large loading rates. PAMAM docked to gp120 and destabilized the gp120-CD4 complex. The rupture force of the complex was lowered by PAMAM. PAMAM disrupted salt bridges and hydrogen bonds across the gp120-CD4 interface and altered the hydration pattern of the hydrophobic cavity in the interface. In addition, intriguingly, PAMAM suppressed the distinction in the dissociation pathways of the complex between the small and large loading rate regimes. Taken together, our simulations reveal that PAMAM targets the gp120-CD4 complex at two levels: it weakens the complex and also alters its dissociation pathway, potentially inhibiting HIV-1 entry.

  18. Simulations reveal that the HIV-1 gp120-CD4 complex dissociates via complex pathways and is a potential target of the polyamidoamine (PAMAM) dendrimer.

    PubMed

    Nandy, Bidisha; Bindu, D Hima; Dixit, Narendra M; Maiti, Prabal K

    2013-07-14

    The polyamidoamine (PAMAM) dendrimer prevents HIV-1 entry into target cells in vitro. Its mechanism of action, however, remains unclear and precludes the design of potent dendrimers targeting HIV-1 entry. We employed steered molecular dynamics simulations to examine whether the HIV-1 gp120-CD4 complex is a target of PAMAM. Our simulations mimicked single molecule force spectroscopy studies of the unbinding of the gp120-CD4 complex under the influence of a controlled external force. We found that the complex dissociates via complex pathways and defies the standard classification of adhesion molecules as catch and slip bonds. When the force loading rate was large, the complex behaved as a slip bond, weakening gradually. When the loading rate was small, the complex initially strengthened, akin to a catch bond, but eventually dissociated over shorter separations than with large loading rates. PAMAM docked to gp120 and destabilized the gp120-CD4 complex. The rupture force of the complex was lowered by PAMAM. PAMAM disrupted salt bridges and hydrogen bonds across the gp120-CD4 interface and altered the hydration pattern of the hydrophobic cavity in the interface. In addition, intriguingly, PAMAM suppressed the distinction in the dissociation pathways of the complex between the small and large loading rate regimes. Taken together, our simulations reveal that PAMAM targets the gp120-CD4 complex at two levels: it weakens the complex and also alters its dissociation pathway, potentially inhibiting HIV-1 entry. PMID:23862963

  19. Unexpected Outcomes from Teachers' TV

    ERIC Educational Resources Information Center

    Tanner, Ruth

    2006-01-01

    In this article, the author explains why she has unexpectedly become a fan of Teacher's TV. It started when she was asked to show some clips from Teachers' TV as part of a workshop she was leading. She dutifully accepted a CD containing three downloaded programmes about using ICT to teach mathematics and put it into her laptop. Within a few…

  20. Wilderness Emergency: Surviving the Unexpected.

    ERIC Educational Resources Information Center

    Fear, Gene

    In any unexpected survival experience, one must accept the situation with just what one has at the moment it happens, where it happens, and how it happens. Problem solving must be based on known body enemies that threaten life, their priority of influence, and their severity of threat to life. Solutions will depend on the body's energy supply,…

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

    NASA Astrophysics Data System (ADS)

    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

  2. Genome Evolution in the Eremothecium Clade of the Saccharomyces Complex Revealed by Comparative Genomics

    PubMed Central

    Wendland, Jürgen; Walther, Andrea

    2011-01-01

    We used comparative genomics to elucidate the genome evolution within the pre–whole-genome duplication genus Eremothecium. To this end, we sequenced and assembled the complete genome of Eremothecium cymbalariae, a filamentous ascomycete representing the Eremothecium type strain. Genome annotation indicated 4712 gene models and 143 tRNAs. We compared the E. cymbalariae genome with that of its relative, the riboflavin overproducer Ashbya (Eremothecium) gossypii, and the reconstructed yeast ancestor. Decisive changes in the Eremothecium lineage leading to the evolution of the A. gossypii genome include the reduction from eight to seven chromosomes, the downsizing of the genome by removal of 10% or 900 kb of DNA, mostly in intergenic regions, the loss of a TY3-Gypsy–type transposable element, the re-arrangement of mating-type loci, and a massive increase of its GC content. Key species-specific events are the loss of MNN1-family of mannosyltransferases required to add the terminal fourth and fifth α-1,3-linked mannose residue to O-linked glycans and genes of the Ehrlich pathway in E. cymbalariae and the loss of ZMM-family of meiosis-specific proteins and acquisition of riboflavin overproduction in A. gossypii. This reveals that within the Saccharomyces complex genome, evolution is not only based on genome duplication with subsequent gene deletions and chromosomal rearrangements but also on fungi associated with specific environments (e.g. involving fungal-insect interactions as in Eremothecium), which have encountered challenges that may be reflected both in genome streamlining and their biosynthetic potential. PMID:22384365

  3. Genome evolution in the eremothecium clade of the Saccharomyces complex revealed by comparative genomics.

    PubMed

    Wendland, Jürgen; Walther, Andrea

    2011-12-01

    We used comparative genomics to elucidate the genome evolution within the pre-whole-genome duplication genus Eremothecium. To this end, we sequenced and assembled the complete genome of Eremothecium cymbalariae, a filamentous ascomycete representing the Eremothecium type strain. Genome annotation indicated 4712 gene models and 143 tRNAs. We compared the E. cymbalariae genome with that of its relative, the riboflavin overproducer Ashbya (Eremothecium) gossypii, and the reconstructed yeast ancestor. Decisive changes in the Eremothecium lineage leading to the evolution of the A. gossypii genome include the reduction from eight to seven chromosomes, the downsizing of the genome by removal of 10% or 900 kb of DNA, mostly in intergenic regions, the loss of a TY3-Gypsy-type transposable element, the re-arrangement of mating-type loci, and a massive increase of its GC content. Key species-specific events are the loss of MNN1-family of mannosyltransferases required to add the terminal fourth and fifth α-1,3-linked mannose residue to O-linked glycans and genes of the Ehrlich pathway in E. cymbalariae and the loss of ZMM-family of meiosis-specific proteins and acquisition of riboflavin overproduction in A. gossypii. This reveals that within the Saccharomyces complex genome, evolution is not only based on genome duplication with subsequent gene deletions and chromosomal rearrangements but also on fungi associated with specific environments (e.g. involving fungal-insect interactions as in Eremothecium), which have encountered challenges that may be reflected both in genome streamlining and their biosynthetic potential. PMID:22384365

  4. Chemical genetics analysis of an aniline mustard anticancer agent reveals complex I of the electron transport chain as a target.

    PubMed

    Fedeles, Bogdan I; Zhu, Angela Y; Young, Kellie S; Hillier, Shawn M; Proffitt, Kyle D; Essigmann, John M; Croy, Robert G

    2011-09-30

    The antitumor agent 11β (CAS 865070-37-7), consisting of a DNA-damaging aniline mustard linked to an androgen receptor (AR) ligand, is known to form covalent DNA adducts and to induce apoptosis potently in AR-positive prostate cancer cells in vitro; it also strongly prevents growth of LNCaP xenografts in mice. The present study describes the unexpectedly strong activity of 11β against the AR-negative HeLa cells, both in cell culture and tumor xenografts, and uncovers a new mechanism of action that likely explains this activity. Cellular fractionation experiments indicated that mitochondria are the major intracellular sink for 11β; flow cytometry studies showed that 11β exposure rapidly induced oxidative stress, mitochondria being an important source of reactive oxygen species (ROS). Additionally, 11β inhibited oxygen consumption both in intact HeLa cells and in isolated mitochondria. Specifically, 11β blocked uncoupled oxygen consumption when mitochondria were incubated with complex I substrates, but it had no effect on oxygen consumption driven by substrates acting downstream of complex I in the mitochondrial electron transport chain. Moreover, 11β enhanced ROS generation in isolated mitochondria, suggesting that complex I inhibition is responsible for ROS production. At the cellular level, the presence of antioxidants (N-acetylcysteine or vitamin E) significantly reduced the toxicity of 11β, implicating ROS production as an important contributor to cytotoxicity. Collectively, our findings establish complex I inhibition and ROS generation as a new mechanism of action for 11β, which supplements conventional DNA adduct formation to promote cancer cell death. PMID:21832047

  5. Structure and flexibility of the endosomal Vps34 complex reveals the basis of its function on membranes

    PubMed Central

    Ohashi, Yohei; Zhang, Lufei; Pardon, Els; Burke, John E.; Masson, Glenn R.; Johnson, Chris; Steyaert, Jan; Ktistakis, Nicholas T.; Williams, Roger L.

    2015-01-01

    Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane trafficking in endocytic sorting, cytokinesis and autophagy. We present the 4.4 Å crystal structure of the 385 kDa endosomal complex II (PIK3C3-CII), consisting of Vps34, Vps15 (p150), Vps30/Atg6 (Beclin 1) and Vps38 (UVRAG). The subunits form a Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one arm where the Vps15 kinase domain engages the Vps34 activation loop to regulate its activity. Vps30 and Vps38 form the other arm that brackets the Vps15/Vps34 heterodimer, suggesting a path for complex assembly. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) revealed conformational changes accompanying membrane binding and identified a Vps30 loop that is critical for the ability of complex II to phosphorylate giant liposomes on which complex I is inactive. PMID:26450213

  6. The Structure of the Yeast Plasma Membrane SNARE Complex Reveals Destabilizing Water Filled Cavities

    SciTech Connect

    Strop, P.; Kaiser, S.E.; Vrljic, M.; Brunger, A.T.

    2009-05-26

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7{angstrom} resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains water-filled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.

  7. Molecular dynamics of the P450cam-Pdx complex reveals complex stability and novel interface contacts.

    PubMed

    Hollingsworth, Scott A; Poulos, Thomas L

    2015-01-01

    Cytochrome P450cam catalyzes the stereo and regiospecific hydroxylation of camphor to 5-exo-hydroxylcamphor. The two electrons for the oxidation of camphor are provided by putidaredoxin (Pdx), a Fe2 S2 containing protein. Two recent crystal structures of the P450cam-Pdx complex, one solved with the aid of covalent cross-linking and one without, have provided a structural picture of the redox partner interaction. To study the stability of the complex structure and the minor differences between the recent crystal structures, a 100 nanosecond molecular dynamics (MD) simulation of the cross-linked structure, mutated in silico to wild type and the linker molecule removed, was performed. The complex was stable over the course of the simulation though conformational changes including the movement of the C helix of P450cam further toward Pdx allowed for the formation of a number of new contacts at the complex interface that remained stable throughout the simulation. While several minor crystal contacts were lost in the simulation, all major contacts that had been experimentally studied previously were maintained. The equilibrated MD structure contained a mixture of contacts resembling both the cross-linked and noncovalent structures and the newly identified interactions. Finally, the reformation of the P450cam Asp251-Arg186 ion pair in the MD simulation mirrors the ion pair observed in the more promiscuous CYP101D1 and suggests that the Asp251-Arg186 ion pair may be important. PMID:25307478

  8. Death Domain Assembly Mechanism Revealed by Crystal Structure of the Oligomeric PIDDosome Core Complex

    SciTech Connect

    Park,H.; Logette, E.; Raunser, S.; Cuenin, S.; Walz, T.; Tschopp, J.; Wu, H.

    2007-01-01

    Proteins of the death domain (DD) superfamily mediate assembly of oligomeric signaling complexes for the activation of caspases and kinases via unknown mechanisms. Here we report the crystal structure of the PIDD DD and RAIDD DD complex, which forms the core of the caspase-2-activating complex PIDDosome. Although RAIDD DD and PIDD DD are monomers, they assemble into a complex that comprises seven RAIDD DDs and five PIDD DDs. Despite the use of an asymmetric assembly mechanism, all DDs in the complex are in quasi-equivalent environments. The structure provided eight unique asymmetric interfaces, which can be classified into three types. These three types of interactions together cover a majority of the DD surface. Mutagenesis on almost all interfaces leads to disruption of the assembly, resulting in defective caspase-2 activation. The three types of interactions may represent most, if not all, modes of interactions in the DD superfamily for assembling complexes of different stoichiometry.

  9. Death Domain Assembly Mechanism Revealed by Crystal Structure of the Oligomeric PIDDosome Core Complex

    PubMed Central

    Park, Hyun Ho; Logette, Emmanuelle; Raunser, Stefan; Cuenin, Solange; Walz, Thomas; Tschopp, Jurg; Wu, Hao

    2010-01-01

    SUMMARY Proteins of the death domain (DD) superfamily mediate assembly of oligomeric signaling complexes for the activation of caspases and kinases via unknown mechanisms. Here we report the crystal structure of the PIDD DD and RAIDD DD complex, which forms the core of the caspase-2 activating complex PIDDosome. While RAIDD DD and PIDD DD are monomers, they assemble into a complex that comprises seven RAIDD DDs and five PIDD DDs. Despite the use of an asymmetric assembly mechanism, all DDs in the complex are in quasi-equivalent environments. The structure provided eight unique asymmetric interfaces, which can be classified into three types. These three types of interactions together cover a majority of the DD surface. Mutagenesis on almost all interfaces leads to disruption of the assembly resulting in defective caspase-2 activation. The three types of interactions may represent most, if not all, modes of interactions in the DD superfamily for assembling complexes of different stoichiometry. PMID:17289572

  10. Comparative Genomic Analysis Reveals 2-Oxoacid Dehydrogenase Complex Lipoylation Correlation with Aerobiosis in Archaea

    PubMed Central

    Borziak, Kirill; Posner, Mareike G.; Upadhyay, Abhishek; Danson, Michael J.; Bagby, Stefan; Dorus, Steve

    2014-01-01

    Metagenomic analyses have advanced our understanding of ecological microbial diversity, but to what extent can metagenomic data be used to predict the metabolic capacity of difficult-to-study organisms and their abiotic environmental interactions? We tackle this question, using a comparative genomic approach, by considering the molecular basis of aerobiosis within archaea. Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multienzyme complexes (OADHCs), is essential for metabolism in aerobic bacteria and eukarya. Lipoylation is catalysed either by lipoate protein ligase (LplA), which in archaea is typically encoded by two genes (LplA-N and LplA-C), or by a lipoyl(octanoyl) transferase (LipB or LipM) plus a lipoic acid synthetase (LipA). Does the genomic presence of lipoylation and OADHC genes across archaea from diverse habitats correlate with aerobiosis? First, analyses of 11,826 biotin protein ligase (BPL)-LplA-LipB transferase family members and 147 archaeal genomes identified 85 species with lipoylation capabilities and provided support for multiple ancestral acquisitions of lipoylation pathways during archaeal evolution. Second, with the exception of the Sulfolobales order, the majority of species possessing lipoylation systems exclusively retain LplA, or either LipB or LipM, consistent with archaeal genome streamlining. Third, obligate anaerobic archaea display widespread loss of lipoylation and OADHC genes. Conversely, a high level of correspondence is observed between aerobiosis and the presence of LplA/LipB/LipM, LipA and OADHC E2, consistent with the role of lipoylation in aerobic metabolism. This correspondence between OADHC lipoylation capacity and aerobiosis indicates that genomic pathway profiling in archaea is informative and that well characterized pathways may be predictive in relation to abiotic conditions in difficult-to-study extremophiles. Given the highly variable retention of gene repertoires across the archaea

  11. Transcriptome Profiling of Taproot Reveals Complex Regulatory Networks during Taproot Thickening in Radish (Raphanus sativus L.)

    PubMed Central

    Yu, Rugang; Wang, Jing; Xu, Liang; Wang, Yan; Wang, Ronghua; Zhu, Xianwen; Sun, Xiaochuan; Luo, Xiaobo; Xie, Yang; Everlyne, Muleke; Liu, Liwang

    2016-01-01

    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 explore the molecular mechanism 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, 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, respectively. A total of 85,939 transcripts were generated from three libraries, from which 10,450, 12,325, and 7392 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 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 attributed to cell differentiation, division and expansion, which are regulated and promoted by certain specific signal transduction pathways and metabolism processes. These results could provide new insights

  12. Transcriptome Profiling of Taproot Reveals Complex Regulatory Networks during Taproot Thickening in Radish (Raphanus sativus L.).

    PubMed

    Yu, Rugang; Wang, Jing; Xu, Liang; Wang, Yan; Wang, Ronghua; Zhu, Xianwen; Sun, Xiaochuan; Luo, Xiaobo; Xie, Yang; Everlyne, Muleke; Liu, Liwang

    2016-01-01

    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 explore the molecular mechanism 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, 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, respectively. A total of 85,939 transcripts were generated from three libraries, from which 10,450, 12,325, and 7392 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 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 attributed to cell differentiation, division and expansion, which are regulated and promoted by certain specific signal transduction pathways and metabolism processes. These results could provide new insights

  13. Unexpected bismuth concentration profiles in metal-organic vapor phase epitaxy-grown Ga(As{sub 1−x}Bi{sub x})/GaAs superlattices revealed by Z-contrast scanning transmission electron microscopy imaging

    SciTech Connect

    Wood, A. W.; Babcock, S. E.; Guan, Y.; Forghani, K.; Anand, A.; Kuech, T. F.

    2015-03-01

    A set of GaAs{sub 1−x}Bi{sub x}/GaAs multilayer quantum-well structures was deposited by metal-organic vapor phase epitaxy at 390 °C and 420 °C. The precursor fluxes were introduced with the intent of growing discrete and compositionally uniform GaAs{sub 1−x}Bi{sub x} well and GaAs barrier layers in the epitaxial films. High-resolution high-angle annular-dark-field (or “Z-contrast”) scanning transmission electron microscopy imaging revealed concentration profiles that were periodic in the growth direction, but far more complicated in shape than the intended square wave. The observed composition profiles could explain various reports of physical properties measurements that suggest compositional inhomogeneity in GaAs{sub 1−x}Bi{sub x} alloys as they currently are grown.

  14. Single molecule microscopy reveals mechanistic insight into RNA polymerase II preinitiation complex assembly and transcriptional activity

    PubMed Central

    Horn, Abigail E.; Kugel, Jennifer F.; Goodrich, James A.

    2016-01-01

    Transcription by RNA polymerase II (Pol II) is a complex process that requires general transcription factors and Pol II to assemble on DNA into preinitiation complexes that can begin RNA synthesis upon binding of NTPs (nucleoside triphosphate). The pathways by which preinitiation complexes form, and how this impacts transcriptional activity are not completely clear. To address these issues, we developed a single molecule system using TIRF (total internal reflection fluorescence) microscopy and purified human transcription factors, which allows us to visualize transcriptional activity at individual template molecules. We see that stable interactions between polymerase II (Pol II) and a heteroduplex DNA template do not depend on general transcription factors; however, transcriptional activity is highly dependent upon TATA-binding protein, TFIIB and TFIIF. We also found that subsets of general transcription factors and Pol II can form stable complexes that are precursors for functional transcription complexes upon addition of the remaining factors and DNA. Ultimately we found that Pol II, TATA-binding protein, TFIIB and TFIIF can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA. Single molecule studies can be used to learn how different modes of preinitiation complex assembly impact transcriptional activity. PMID:27112574

  15. [Unexpected revision procedures treating ankle fractures].

    PubMed

    Richter, J; Pommer, A; Breuer, R; Hullmann, S; Heyde, D V; Dávid, A

    2012-06-01

    The purpose of the present study was to analyze the risk factors associated with unexpected second procedures and strategies of revision surgery. Within a 5 year period 647 patients with closed ankle fractures AO type 44 were identified of which 77 (11.9%) needed revision surgery. Complications were addressed to 4 main groups: deep infections (IG) were seen in 29 patients (4.5%), problems with primary wound closure (WG) in 22 patients (3.4%), insufficient reduction (KG) in 22 patients (3.4%) and other causes (RG) included 4 patients (0.6%). Significant predictive factors for soft tissue complications were higher age, comorbidities with peripheral arteriosclerosis, high American Society of Anesthesiologists (ASA) score and diabetes mellitus. AO 44 type B2 and B3 fractures were often associated with soft tissue problems. The more complex fracture types AO 44 C1-C3 and A2-A3 were significantly associated with problems of insufficient congruency post-surgery. The distribution of the mean revision rate was significantly different (p<0.01) for all groups: IG 4.59, WG 3.5, KG 1.55, RG 1.25. In summary, we strongly recommend immediate reduction of displaced fractures and to consider a more detailed fracture classification. To reduce the amount of unexpected ankle procedures individual risk factors should be weighed against the advantages of optimal open reduction and internal fixation. PMID:21165587

  16. Paleotopographic Reconstruction of the Tharsis Magmatic Complex Reveals Potential Ancient Drainage Basin/Aquifer System

    NASA Technical Reports Server (NTRS)

    Dohm, J. M.; Ferris, J.; Anderson, R. C.; Baker, V.; Hare, T.; Barlow, N. G.; Strom, R. G.; Tanaka, K. L.; Scott, D. H.

    2001-01-01

    Paleotopographic reconstructions reveal the potential existence of an enormous Noachian drainage basin in the eastern part of the Tharsis region of significant geologic and paleohydrologic implications. Additional information is contained in the original extended abstract.

  17. DISTILLER: a data integration framework to reveal condition dependency of complex regulons in Escherichia coli

    PubMed Central

    Lemmens, Karen; De Bie, Tijl; Dhollander, Thomas; De Keersmaecker, Sigrid C; Thijs, Inge M; Schoofs, Geert; De Weerdt, Ami; De Moor, Bart; Vanderleyden, Jos; Collado-Vides, Julio; Engelen, Kristof; Marchal, Kathleen

    2009-01-01

    We present DISTILLER, a data integration framework for the inference of transcriptional module networks. Experimental validation of predicted targets for the well-studied fumarate nitrate reductase regulator showed the effectiveness of our approach in Escherichia coli. In addition, the condition dependency and modularity of the inferred transcriptional network was studied. Surprisingly, the level of regulatory complexity seemed lower than that which would be expected from RegulonDB, indicating that complex regulatory programs tend to decrease the degree of modularity. PMID:19265557

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

    PubMed

    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. PMID:27340174

  19. Rapid Binding of Plasminogen to Streptokinase in a Catalytic Complex Reveals a Three-step Mechanism*

    PubMed Central

    Verhamme, Ingrid M.; Bock, Paul E.

    2014-01-01

    Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys414 deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-d-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys414 deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys414 to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK·Pg complex compared with SK·Pm is characterized by a ∼25-fold weaker encounter complex and ∼40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys414 engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK·Pg complex and the expression of a weaker “pro”-exosite for binding of a second Pg in the substrate mode compared with SK·Pm. PMID:25138220

  20. Rapid binding of plasminogen to streptokinase in a catalytic complex reveals a three-step mechanism.

    PubMed

    Verhamme, Ingrid M; Bock, Paul E

    2014-10-01

    Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys(414) deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-D-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK ·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys(414) deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys(414) to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK · Pg complex compared with SK · Pm is characterized by a ∼ 25-fold weaker encounter complex and ∼ 40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys(414) engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK · Pg complex and the expression of a weaker "pro"-exosite for binding of a second Pg in the substrate mode compared with SK · Pm. PMID:25138220

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

    SciTech Connect

    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.

  2. A method for multiprotein assembly in cells reveals independent action of kinesins in complex

    PubMed Central

    Norris, Stephen R.; Soppina, Virupakshi; Dizaji, Aslan S.; Schimert, Kristin I.; Sept, David; Cai, Dawen; Sivaramakrishnan, Sivaraj

    2014-01-01

    Teams of processive molecular motors are critical for intracellular transport and organization, yet coordination between motors remains poorly understood. Here, we develop a system using protein components to generate assemblies of defined spacing and composition inside cells. This system is applicable to studying macromolecular complexes in the context of cell signaling, motility, and intracellular trafficking. We use the system to study the emergent behavior of kinesin motors in teams. We find that two kinesin motors in complex act independently (do not help or hinder each other) and can alternate their activities. For complexes containing a slow kinesin-1 and fast kinesin-3 motor, the slow motor dominates motility in vitro but the fast motor can dominate on certain subpopulations of microtubules in cells. Both motors showed dynamic interactions with the complex, suggesting that motor–cargo linkages are sensitive to forces applied by the motors. We conclude that kinesin motors in complex act independently in a manner regulated by the microtubule track. PMID:25365993

  3. Impedance technology reveals correlations between cytotoxicity and lipophilicity of mono and bimetallic phosphine complexes.

    PubMed

    Fonteh, P; Elkhadir, A; Omondi, B; Guzei, I; Darkwa, J; Meyer, D

    2015-08-01

    Label free impedance technology enables the monitoring of cell response patterns post treatment with drugs or other chemicals. Using this technology, a correlation between the lipophilicity of metal complexes and the degree of cytotoxicity was observed. Au(L1)Cl (1), AuPd(L1)(SC4H8)Cl3 (1a) and Au(L2)Cl (2) [L1 = diphenylphosphino-2-pyridine; L2 = 2-(2-(diphenylphosphino)ethyl)-pyridine] were synthesised, in silico drug-likeness and structure-activity relationship monitored using impedance technology. Dose dependent changes in cytotoxicity were observed for the metal complexes resulting in IC50s of 12.5 ± 2.5, 18.3 ± 8.3 and 16.9 ± 0.5 µM for 1, 1a and 2 respectively in an endpoint assay. When a real time impedance assay was used, dose-dependent responses depicting patterns that suggested slower uptake (at a toxic 20 µM) and faster recovery of the cells (at the less toxic 10 µM) of the bimetallic complex (1a) compared to the monometallic complexes (1 and 2) was observed. These data agreed with the ADMET findings of lower aqueous solubility of 1a and non-ideal lipophilicity (AlogP98 of 6.55) over more water soluble 1 and 2 with ideal lipophilicity (4.91 and 5.03 respectively) values. The additional coordination of a Pd atom to the nitrogen atom of a pyridine ring, the sulfur atom of a tetrahydrothiophene moiety and two chlorine atoms in 1a could be contributing to the observed differences when compared to the monometallic complexes. This report presents impedance technology as a means of correlating drug-likeness of lipophilic phosphine complexes containing similar backbone structures and could prove valuable in filtering drug-like compounds in a drug discovery project. PMID:25829148

  4. The comet-like composition of a protoplanetary disk as revealed by complex cyanides.

    PubMed

    Öberg, Karin I; Guzmán, Viviana V; Furuya, Kenji; Qi, Chunhua; Aikawa, Yuri; Andrews, Sean M; Loomis, Ryan; Wilner, David J

    2015-04-01

    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. Unlike asteroids, comets preserve a nearly pristine record of the solar nebula composition. The presence of cyanides in comets, including 0.01 per cent 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 readily be explained by a combination of gas-phase chemistry (to form, for example, HCN) and an active ice-phase chemistry on grain surfaces that advances complexity. Simple volatiles, including water and HCN, have been detected previously in solar nebula analogues, indicating that they survive disk formation or are re-formed in situ. It has hitherto been unclear whether the same holds for more complex organic molecules outside the solar nebula, given that recent observations show a marked change in the chemistry at the boundary between nascent envelopes and young disks due to accretion shocks. Here we report the detection of the complex cyanides CH3CN and HC3N (and HCN) in the protoplanetary disk around the young star MWC 480. We find that the abundance ratios of these nitrogen-bearing organics in the gas phase are similar to those in comets, which suggests an even higher relative abundance of complex cyanides in the disk ice. This implies that complex organics accompany simpler volatiles in protoplanetary disks, and that the rich organic chemistry of our solar nebula was not unique. PMID:25855455

  5. The comet-like composition of a protoplanetary disk as revealed by complex cyanides

    NASA Astrophysics Data System (ADS)

    Öberg, Karin I.; Guzmán, Viviana V.; Furuya, Kenji; Qi, Chunhua; Aikawa, Yuri; Andrews, Sean M.; Loomis, Ryan; Wilner, David J.

    2015-04-01

    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. Unlike asteroids, comets preserve a nearly pristine record of the solar nebula composition. The presence of cyanides in comets, including 0.01 per cent 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 readily be explained by a combination of gas-phase chemistry (to form, for example, HCN) and an active ice-phase chemistry on grain surfaces that advances complexity. Simple volatiles, including water and HCN, have been detected previously in solar nebula analogues, indicating that they survive disk formation or are re-formed in situ. It has hitherto been unclear whether the same holds for more complex organic molecules outside the solar nebula, given that recent observations show a marked change in the chemistry at the boundary between nascent envelopes and young disks due to accretion shocks. Here we report the detection of the complex cyanides CH3CN and HC3N (and HCN) in the protoplanetary disk around the young star MWC 480. We find that the abundance ratios of these nitrogen-bearing organics in the gas phase are similar to those in comets, which suggests an even higher relative abundance of complex cyanides in the disk ice. This implies that complex organics accompany simpler volatiles in protoplanetary disks, and that the rich organic chemistry of our solar nebula was not unique.

  6. Proteomics Analysis with a Nano Random Forest Approach Reveals Novel Functional Interactions Regulated by SMC Complexes on Mitotic Chromosomes*

    PubMed Central

    Ohta, Shinya; Montaño-Gutierrez, Luis F.; de Lima Alves, Flavia; Ogawa, Hiromi; Toramoto, Iyo; Sato, Nobuko; Morrison, Ciaran G.; Takeda, Shunichi; Hudson, Damien F.; Earnshaw, William C.

    2016-01-01

    Packaging of DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although the structure and composition of mitotic chromosomes have been studied for over 30 years, these aspects are yet to be fully elucidated. Here, we used stable isotope labeling with amino acids in cell culture to compare the proteomes of mitotic chromosomes isolated from cell lines harboring conditional knockouts of members of the condensin (SMC2, CAP-H, CAP-D3), cohesin (Scc1/Rad21), and SMC5/6 (SMC5) complexes. Our analysis revealed that these complexes associate with chromosomes independently of each other, with the SMC5/6 complex showing no significant dependence on any other chromosomal proteins during mitosis. To identify subtle relationships between chromosomal proteins, we employed a nano Random Forest (nanoRF) approach to detect protein complexes and the relationships between them. Our nanoRF results suggested that as few as 113 of 5058 detected chromosomal proteins are functionally linked to chromosome structure and segregation. Furthermore, nanoRF data revealed 23 proteins that were not previously suspected to have functional interactions with complexes playing important roles in mitosis. Subsequent small-interfering-RNA-based validation and localization tracking by green fluorescent protein-tagging highlighted novel candidates that might play significant roles in mitotic progression. PMID:27231315

  7. Proteomics Analysis with a Nano Random Forest Approach Reveals Novel Functional Interactions Regulated by SMC Complexes on Mitotic Chromosomes.

    PubMed

    Ohta, Shinya; Montaño-Gutierrez, Luis F; de Lima Alves, Flavia; Ogawa, Hiromi; Toramoto, Iyo; Sato, Nobuko; Morrison, Ciaran G; Takeda, Shunichi; Hudson, Damien F; Rappsilber, Juri; Earnshaw, William C

    2016-08-01

    Packaging of DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although the structure and composition of mitotic chromosomes have been studied for over 30 years, these aspects are yet to be fully elucidated. Here, we used stable isotope labeling with amino acids in cell culture to compare the proteomes of mitotic chromosomes isolated from cell lines harboring conditional knockouts of members of the condensin (SMC2, CAP-H, CAP-D3), cohesin (Scc1/Rad21), and SMC5/6 (SMC5) complexes. Our analysis revealed that these complexes associate with chromosomes independently of each other, with the SMC5/6 complex showing no significant dependence on any other chromosomal proteins during mitosis. To identify subtle relationships between chromosomal proteins, we employed a nano Random Forest (nanoRF) approach to detect protein complexes and the relationships between them. Our nanoRF results suggested that as few as 113 of 5058 detected chromosomal proteins are functionally linked to chromosome structure and segregation. Furthermore, nanoRF data revealed 23 proteins that were not previously suspected to have functional interactions with complexes playing important roles in mitosis. Subsequent small-interfering-RNA-based validation and localization tracking by green fluorescent protein-tagging highlighted novel candidates that might play significant roles in mitotic progression. PMID:27231315

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

    SciTech Connect

    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.

  9. Stoichiometry of a regulatory splicing complex revealed by single-molecule analyses.

    PubMed

    Cherny, Dmitry; Gooding, Clare; Eperon, Giles E; Coelho, Miguel B; Bagshaw, Clive R; Smith, Christopher W J; Eperon, Ian C

    2010-07-01

    Splicing is regulated by complex interactions of numerous RNA-binding proteins. The molecular mechanisms involved remain elusive, in large part because of ignorance regarding the numbers of proteins in regulatory complexes. Polypyrimidine tract-binding protein (PTB), which regulates tissue-specific splicing, represses exon 3 of alpha-tropomyosin through distant pyrimidine-rich tracts in the flanking introns. Current models for repression involve either PTB-mediated looping or the propagation of complexes between tracts. To test these models, we used single-molecule approaches to count the number of bound PTB molecules both by counting the number of bleaching steps of GFP molecules linked to PTB within complexes and by analysing their total emissions. Both approaches showed that five or six PTB molecules assemble. Given the domain structures, this suggests that the molecules occupy primarily multiple overlapping potential sites in the polypyrimidine tracts, excluding propagation models. As an alternative to direct looping, we propose that repression involves a multistep process in which PTB binding forms small local loops, creating a platform for recruitment of other proteins that bring these loops into close proximity. PMID:20502437

  10. Unprecedented staining of polar lipids by a luminescent rhenium complex revealed by FTIR microspectroscopy in adipocytes.

    PubMed

    Bader, C A; Carter, E A; Safitri, A; Simpson, P V; Wright, P; Stagni, S; Massi, M; Lay, P A; Brooks, D A; Plush, S E

    2016-06-21

    Fourier transform infrared (FTIR) microspectroscopy and confocal imaging have been used to demonstrate that the neutral rhenium(i) tricarbonyl 1,10-phenanthroline complex bound to 4-cyanophenyltetrazolate as the ancillary ligand is able to localise in regions with high concentrations of polar lipids such as phosphatidylethanolamine (PE), sphingomyelin, sphingosphine and lysophosphatidic acid (LPA) in mammalian adipocytes. PMID:27170554

  11. Behaviour of Zinc Complexes and Zinc Sulphide Nanoparticles Revealed by Using Screen Printed Electrodes and Spectrometry

    PubMed Central

    Nejdl, Lukas; Ruttkay-Nedecky, Branislav; Kudr, Jiří; Kremplova, Monika; Cernei, Natalia; Prasek, Jan; Konecna, Marie; Hubalek, Jaromir; Zitka, Ondrej; Kynicky, Jindrich; Kopel, Pavel; Kizek, Rene; Adam, Vojtech

    2013-01-01

    In this study, we focused on microfluidic electrochemical analysis of zinc complexes (Zn(phen)(his)Cl2, Zn(his)Cl2) and ZnS quantum dots (QDs) using printed electrodes. This method was chosen due to the simple (easy to use) instrumentation and variable setting of flows. Reduction signals of zinc under the strictly defined and controlled conditions (pH, temperature, flow rate, accumulation time and applied potential) were studied. We showed that the increasing concentration of the complexes (Zn(phen)(his)Cl2, Zn(his)Cl2) led to a decrease in the electrochemical signal and a significant shift of the potential to more positive values. The most likely explanation of this result is that zinc is strongly bound in the complex and its distribution on the electrode is very limited. Changing the pH from 3.5 to 5.5 resulted in a significant intensification of the Zn(II) reduction signal. The complexes were also characterized by UV/VIS spectrophotometry, chromatography, and ESI-QTOF mass spectrometry. PMID:24233071

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

    ERIC Educational Resources Information Center

    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. Asymmetric collapse in biomimetic complex coacervates revealed by local polymer and water dynamics.

    PubMed

    Ortony, Julia H; Hwang, Dong Soo; Franck, John M; Waite, J Herbert; Han, Songi

    2013-05-13

    Complex coacervation is a phenomenon characterized by the association of oppositely charged polyelectrolytes into micrometer-scale liquid condensates. This process is the purported first step in the formation of underwater adhesives by sessile marine organisms, as well as the process harnessed for the formation of new synthetic and protein-based contemporary materials. Efforts to understand the physical nature of complex coacervates are important for developing robust adhesives, injectable materials, or novel drug delivery vehicles for biomedical applications; however, their internal fluidity necessitates the use of in situ characterization strategies of their local dynamic properties, capabilities not offered by conventional techniques such as X-ray scattering, microscopy, or bulk rheological measurements. Herein, we employ the novel magnetic resonance technique Overhauser dynamic nuclear polarization enhanced nuclear magnetic resonance (DNP), together with electron paramagnetic resonance (EPR) line shape analysis, to concurrently quantify local molecular and hydration dynamics, with species- and site-specificity. We observe striking differences in the structure and dynamics of the protein-based biomimetic complex coacervates from their synthetic analogues, which is an asymmetric collapse of the polyelectrolyte constituents. From this study we suggest charge heterogeneity within a given polyelectrolyte chain to be an important parameter by which the internal structure of complex coacervates may be tuned. Acquiring molecular-level insight to the internal structure and dynamics of dynamic polymer complexes in water through the in situ characterization of site- and species-specific local polymer and hydration dynamics should be a promising general approach that has not been widely employed for materials characterization. PMID:23540713

  14. Complex history of admixture during citrus domestication revealed by genome analysis

    SciTech Connect

    Wu, G. Albert; Prochnik, Simon; Jenkins, Jerry; Salse, Jerome; Hellsten, Uffe; Murat, Florent; Perrier, Xavier; Ruiz, Manuel; Scalabrin, Simone; Terol, Javier; Takita, Marco Auré lio,; Labadie, Karine; Poulain, Julie; Couloux, Arnaud; Jabbari, Kamel; Cattonaro, Federica; Fabbro, Cristian Del; Pinosio, Sara; Zuccolo, Andrea; Chapman, Jarrod; Grimwood, Jane; Tadeo, Francisco; Estornell, Leandro H.; Mu?oz-Sanz, Juan V.; Ibanez, Victoria; Herrero-Ortega, Amparo; Aleza, Pablo; Pé rez, Juliá n Pé rez,; Ramon, Daniel; Brunel, Dominique; Luro, Francois; Chen, Chunxian; Farmerie, William G.; Desany, Brian; Kodira, Chinnappa; Mohiuddin, Mohammed; Harkins, Tim; Fredrikson, Karin; Burns, Paul; Lomsadze, Alexandre; Borodovsky, Mark; Reforgiato, Giuseppe; Freitas-Astua, Juliana; Quetier, Francis; Navarro, Luis; Roose, Mikeal; Wincker, Patrick; Schmutz, Jeremy; Morgante, Michele; Machado, Marcos Antonio; Talon, Manuel; Jaillon, Olivier; Ollitrault, Patrick; Gmitter, Frederick; Rokhsar, Daniel

    2014-06-30

    Although Citrus is the most globally significant tree fruit, its domestication history is poorly understood. Cultivated citrus types are believed to comprise selections from and/or hybrids of several wild progenitor species, but the identities of these progenitors, and their contribution to modern cultivars, remain controversial. Here we report the genomes of a collection of mandarins, pummelos, and oranges, including a high quality reference sequence from a haploid Clementine mandarin. By comparative genome analysis we show that these cultivated types can be derived from two progenitor species. Cultivated pummelos represent selections from a single progenitor species C. maxima. Unexpectedly, however, we find that cultivated mandarins are introgressions of C. maxima into a distinct second population that we identify with the ancestral wild mandarin species C. reticulata. Sweet and sour oranges are found to be interspecific hybrids. Sweet orange, the most widely cultivated citrus, arose as the offspring of previously admixed individuals. In contrast, sour (or Seville) orange is an F1 hybrid of pure C. maxima and C. reticulata parents, implying that wild mandarins were part of the early breeding germplasm. Surprisingly, we also find that a wild Chinese mandarin from Mangshan, China shows substantial sequence divergence from C. reticulata and appears to represent a distinct taxon. Understanding the relationships and phylogeny of cultivated citrus through genome analysis will clarify taxonomic relationships and enable previously inconceivable opportunities for sequence-directed genetic improvement. Citrus are widely consumed worldwide as juice or fresh fruit, providing important sources of vitamin C and other health-promoting compounds. Global production in 2012 exceeded 86 million metric tons, with an estimated value of US$9 billion (http://www.fas.usda.gov/psdonline/circulars/citrus.pdf). The very narrow genetic diversity of cultivated citrus makes it highly

  15. Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction

    PubMed Central

    Laxmikanthan, Gurunathan; Xu, Chen; Brilot, Axel F; Warren, David; Steele, Lindsay; Seah, Nicole; Tong, Wenjun; Grigorieff, Nikolaus; Landy, Arthur; Van Duyne, Gregory D

    2016-01-01

    The molecular machinery responsible for DNA expression, recombination, and compaction has been difficult to visualize as functionally complete entities due to their combinatorial and structural complexity. We report here the structure of the intact functional assembly responsible for regulating and executing a site-specific DNA recombination reaction. The assembly is a 240-bp Holliday junction (HJ) bound specifically by 11 protein subunits. This higher-order complex is a key intermediate in the tightly regulated pathway for the excision of bacteriophage λ viral DNA out of the E. coli host chromosome, an extensively studied paradigmatic model system for the regulated rearrangement of DNA. Our results provide a structural basis for pre-existing data describing the excisive and integrative recombination pathways, and they help explain their regulation. DOI: http://dx.doi.org/10.7554/eLife.14313.001 PMID:27223329

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

    SciTech Connect

    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. Lastly, this method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes.

  17. Gas-phase unfolding and disassembly reveals stability differences in ligand-bound multiprotein complexes.

    PubMed

    Hyung, Suk-Joon; Robinson, Carol V; Ruotolo, Brandon T

    2009-04-24

    Mass spectrometry (MS) is widely used to assess the binding of small molecules to proteins and their complexes. In many cases, subtle differences in the stability afforded by binding of ligands to protein assemblies cannot be detected by MS. Here we show that monitoring the unfolding of protein subunits, using ion mobility-MS, allows differentiation of the effects of ligand binding not normally observed by MS alone. Using wild-type and disease-associated variants of tetrameric transthyretin, MS data indicate that populations of the variant protein are less stable than wild-type. Ion mobility-MS, however, is able to show that the natural ligand of transthyretin, thyroxine, provides a larger stability increase to the tetramer composed of variant subunits than to the wild-type protein-ligand complex. Overall, therefore, our results have implications for small-molecule drug design directed at multiprotein targets. PMID:19389624

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

    SciTech Connect

    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

    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. Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction.

    PubMed

    Laxmikanthan, Gurunathan; Xu, Chen; Brilot, Axel F; Warren, David; Steele, Lindsay; Seah, Nicole; Tong, Wenjun; Grigorieff, Nikolaus; Landy, Arthur; Van Duyne, Gregory D

    2016-01-01

    The molecular machinery responsible for DNA expression, recombination, and compaction has been difficult to visualize as functionally complete entities due to their combinatorial and structural complexity. We report here the structure of the intact functional assembly responsible for regulating and executing a site-specific DNA recombination reaction. The assembly is a 240-bp Holliday junction (HJ) bound specifically by 11 protein subunits. This higher-order complex is a key intermediate in the tightly regulated pathway for the excision of bacteriophage λ viral DNA out of the E. coli host chromosome, an extensively studied paradigmatic model system for the regulated rearrangement of DNA. Our results provide a structural basis for pre-existing data describing the excisive and integrative recombination pathways, and they help explain their regulation. PMID:27223329

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

    PubMed

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

    2015-01-01

    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. PMID:26119246

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26119246

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

    DOE PAGESBeta

    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. Lastly,more » this method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes.« less

  4. Electron Microscopy Analysis of a Disaccharide Analog complex Reveals Receptor Interactions of Adeno-Associated Virus

    PubMed Central

    Xie, Qing; Spilman, Michael; Meyer, Nancy L.; Lerch, Thomas F.; Stagg, Scott M.; Chapman, Michael S.

    2013-01-01

    Mechanistic studies of macromolecular complexes often feature x-ray structures of complexes with bound ligands. The attachment of Adeno-Associated Virus (AAV) to cell surface glycosaminoglycans (GAGs) is an example that has not proven amenable to crystallography, because the binding of GAG analogs disrupts lattice contacts. The interactions of AAV with GAGs are of interest in mediating the cell specificity of AAV-based gene therapy vectors. Previous electron microscopy led to differing conclusions on the exact binding site and the existence of large ligand-induced conformational changes in the virus. Conformational changes are expected during cell entry, but it has remained unclear whether the electron microscopy provided evidence of their induction by GAG-binding. Taking advantage of automated data collection, careful processing and new methods of structure refinement, the structure of AAV-DJ complexed with sucrose octasulfate is determined by electron microscopy difference map analysis to 4.8 Å resolution. At this higher resolution, individual sulfate groups are discernible, providing a stereochemical validation of map interpretation, and highlighting interactions with two surface arginines that have been implicated in genetic studies. Conformational changes induced by the SOS are modest and limited to the loop most directly interacting with the ligand. While the resolution attainable will depend on sample order and other factors, there are an increasing number of macromolecular complexes that can be studied by cryo-electron microscopy at resolutions beyond 5 Å, for which the approaches used here could be used to characterize the binding of inhibitors and other small molecule effectors when crystallography is not tractable. PMID:24036405

  5. Test of Colonisation Scenarios Reveals Complex Invasion History of the Red Tomato Spider Mite Tetranychus evansi

    PubMed Central

    Roderick, George K.; Auger, Philippe; Cornuet, Jean-Marie; Magalhães, Sara; Navajas, Maria

    2012-01-01

    The spider mite Tetranychus evansi is an emerging pest of solanaceous crops worldwide. Like many other emerging pests, its small size, confusing taxonomy, complex history of associations with humans, and propensity to start new populations from small inocula, make the study of its invasion biology difficult. Here, we use recent developments in Approximate Bayesian Computation (ABC) and variation in multi-locus genetic markers to reconstruct the complex historical demography of this cryptic invasive pest. By distinguishing among multiple pathways and timing of introductions, we find evidence for the “bridgehead effect”, in which one invasion serves as source for subsequent invasions. Tetranychus evansi populations in Europe and Africa resulted from at least three independent introductions from South America and involved mites from two distinct sources in Brazil, corresponding to highly divergent mitochondrial DNA lineages. Mites from southwest Brazil (BR-SW) colonized the African continent, and from there Europe through two pathways in a “bridgehead” type pattern. One pathway resulted in a widespread invasion, not only to Europe, but also to other regions in Africa, southern Europe and eastern Asia. The second pathway involved the mixture with a second introduction from BR-SW leading to an admixed population in southern Spain. Admixture was also detected between invasive populations in Portugal. A third introduction from the Brazilian Atlantic region resulted in only a limited invasion in Europe. This study illustrates that ABC methods can provide insights into, and distinguish among, complex invasion scenarios. These processes are critical not only in understanding the biology of invasions, but also in refining management strategies for invasive species. For example, while reported observations of the mite and outbreaks in the invaded areas were largely consistent with estimates of geographical expansion from the ABC approach, historical observations failed

  6. Two-dimensional infrared spectroscopy reveals the complex behaviour of an amyloid fibril inhibitor

    NASA Astrophysics Data System (ADS)

    Middleton, Chris T.; Marek, Peter; Cao, Ping; Chiu, Chi-Cheng; Singh, Sadanand; Woys, Ann Marie; de Pablo, Juan J.; Raleigh, Daniel P.; Zanni, Martin T.

    2012-05-01

    Amyloid formation has been implicated in the pathology of over 20 human diseases, but the rational design of amyloid inhibitors is hampered by a lack of structural information about amyloid-inhibitor complexes. We use isotope labelling and two-dimensional infrared spectroscopy to obtain a residue-specific structure for the complex of human amylin (the peptide responsible for islet amyloid formation in type 2 diabetes) with a known inhibitor (rat amylin). Based on its sequence, rat amylin should block formation of the C-terminal β-sheet, but at 8 h after mixing, rat amylin blocks the N-terminal β-sheet instead. At 24 h after mixing, rat amylin blocks neither β-sheet and forms its own β-sheet, most probably on the outside of the human fibrils. This is striking, because rat amylin is natively disordered and not previously known to form amyloid β-sheets. The results show that even seemingly intuitive inhibitors may function by unforeseen and complex structural processes.

  7. Cysteine scanning reveals minor local rearrangements of the horizontal helix of respiratory complex I.

    PubMed

    Steimle, Stefan; Schnick, Christian; Burger, Eva-Maria; Nuber, Franziska; Krämer, Dorothée; Dawitz, Hannah; Brander, Sofia; Matlosz, Bartlomiej; Schäfer, Jacob; Maurer, Katharina; Glessner, Udo; Friedrich, Thorsten

    2015-10-01

    The NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the redox reaction and a membrane arm catalyzing proton translocation. The membrane arm is almost completely aligned by a 110 Å unique horizontal helix that is discussed to transmit conformational changes induced by the redox reaction in a piston-like movement to the membrane arm driving proton translocation. Here, we analyzed such a proposed movement by cysteine-scanning of the helix of the Escherichia coli complex I. The accessibility of engineered cysteine residues and the flexibility of individual positions were determined by labeling the preparations with a fluorescent marker and a spin-probe, respectively, in the oxidized and reduced states. The differences in fluorescence labeling and the rotational flexibility of the spin probe between both redox states indicate only slight conformational changes at distinct positions of the helix but not a large movement. PMID:26115017

  8. The solution structure of a specific GAGA factor-DNA complex reveals a modular binding mode.

    PubMed

    Omichinski, J G; Pedone, P V; Felsenfeld, G; Gronenborn, A M; Clore, G M

    1997-02-01

    The structure of a complex between the DNA binding domain of the GAGA factor (GAGA-DBD) and an oligonucleotide containing its GAGAG consensus binding site has been determined by nuclear magnetic resonance spectroscopy. The GAGA-DBD comprises a single classical Cys2-His2 zinc finger core, and an N-terminal extension containing two highly basic regions, BR1 and BR2. The zinc finger core binds in the major groove and recognizes the first three GAG bases of the consensus in a manner similar to that seen in other classical zinc finger-DNA complexes. Unlike the latter, which require tandem zinc finger repeats with a minimum of two units for high affinity binding, the GAGA-DBD makes use of only a single finger complemented by BR1 and BR2. BR2 forms a helix that interacts in the major groove recognizing the last G of the consensus, while BR1 wraps around the DNA in the minor groove and recognizes the A in the fourth position of the consensus. The implications of the structure of the GAGA-DBD-DNA complex for chromatin remodelling are discussed. PMID:9033593

  9. Interactome Mapping Reveals the Evolutionary History of the Nuclear Pore Complex

    PubMed Central

    Obado, Samson O.; Brillantes, Marc; Uryu, Kunihiro; Zhang, Wenzhu; Ketaren, Natalia E.; Chait, Brian T.; Field, Mark C.; Rout, Michael P.

    2016-01-01

    The nuclear pore complex (NPC) is responsible for nucleocytoplasmic transport and constitutes a hub for control of gene expression. The components of NPCs from several eukaryotic lineages have been determined, but only the yeast and vertebrate NPCs have been extensively characterized at the quaternary level. Significantly, recent evidence indicates that compositional similarity does not necessarily correspond to homologous architecture between NPCs from different taxa. To address this, we describe the interactome of the trypanosome NPC, a representative, highly divergent eukaryote. We identify numerous new NPC components and report an exhaustive interactome, allowing assignment of trypanosome nucleoporins to discrete NPC substructures. Remarkably, despite retaining similar protein composition, there are exceptional architectural dissimilarities between opisthokont (yeast and vertebrates) and excavate (trypanosomes) NPCs. Whilst elements of the inner core are conserved, numerous peripheral structures are highly divergent, perhaps reflecting requirements to interface with divergent nuclear and cytoplasmic functions. Moreover, the trypanosome NPC has almost complete nucleocytoplasmic symmetry, in contrast to the opisthokont NPC; this may reflect divergence in RNA export processes at the NPC cytoplasmic face, as we find evidence supporting Ran-dependent mRNA export in trypanosomes, similar to protein transport. We propose a model of stepwise acquisition of nucleocytoplasmic mechanistic complexity and demonstrate that detailed dissection of macromolecular complexes provides fuller understanding of evolutionary processes. PMID:26891179

  10. Interactome Mapping Reveals the Evolutionary History of the Nuclear Pore Complex.

    PubMed

    Obado, Samson O; Brillantes, Marc; Uryu, Kunihiro; Zhang, Wenzhu; Ketaren, Natalia E; Chait, Brian T; Field, Mark C; Rout, Michael P

    2016-02-01

    The nuclear pore complex (NPC) is responsible for nucleocytoplasmic transport and constitutes a hub for control of gene expression. The components of NPCs from several eukaryotic lineages have been determined, but only the yeast and vertebrate NPCs have been extensively characterized at the quaternary level. Significantly, recent evidence indicates that compositional similarity does not necessarily correspond to homologous architecture between NPCs from different taxa. To address this, we describe the interactome of the trypanosome NPC, a representative, highly divergent eukaryote. We identify numerous new NPC components and report an exhaustive interactome, allowing assignment of trypanosome nucleoporins to discrete NPC substructures. Remarkably, despite retaining similar protein composition, there are exceptional architectural dissimilarities between opisthokont (yeast and vertebrates) and excavate (trypanosomes) NPCs. Whilst elements of the inner core are conserved, numerous peripheral structures are highly divergent, perhaps reflecting requirements to interface with divergent nuclear and cytoplasmic functions. Moreover, the trypanosome NPC has almost complete nucleocytoplasmic symmetry, in contrast to the opisthokont NPC; this may reflect divergence in RNA export processes at the NPC cytoplasmic face, as we find evidence supporting Ran-dependent mRNA export in trypanosomes, similar to protein transport. We propose a model of stepwise acquisition of nucleocytoplasmic mechanistic complexity and demonstrate that detailed dissection of macromolecular complexes provides fuller understanding of evolutionary processes. PMID:26891179

  11. A novel mode of nuclease action is revealed by the bacterial Mre11/Rad50 complex

    PubMed Central

    Lim, Chew Theng; Lai, Pey Jiun; Leach, David R. F.; Maki, Hisaji; Furukohri, Asako

    2015-01-01

    The Mre11/Rad50 complex is a central player in various genome maintenance pathways. Here, we report a novel mode of nuclease action found for the Escherichia coli Mre11/Rad50 complex, SbcC2/D2 complex (SbcCD). SbcCD cuts off the top of a cruciform DNA by making incisions on both strands and continues cleaving the dsDNA stem at ∼10-bp intervals. Using linear-shaped DNA substrates, we observed that SbcCD cleaved dsDNA using this activity when the substrate was 110 bp long, but that on shorter substrates the cutting pattern was changed to that predicted for the activity of a 3′-5′ exonuclease. Our results suggest that SbcCD processes hairpin and linear dsDNA ends with this novel DNA end-dependent binary endonuclease activity in response to substrate length rather than using previously reported activities. We propose a model for this mode of nuclease action, which provides new insight into SbcCD activity at a dsDNA end. PMID:26319016

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

    PubMed

    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. PMID:26984495

  13. Crystal structures of the CPAP/STIL complex reveal its role in centriole assembly and human microcephaly

    PubMed Central

    Cottee, Matthew A; Muschalik, Nadine; Wong, Yao Liang; Johnson, Christopher M; Johnson, Steven; Andreeva, Antonina; Oegema, Karen; Lea, Susan M; Raff, Jordan W; van Breugel, Mark

    2013-01-01

    Centrioles organise centrosomes and template cilia and flagella. Several centriole and centrosome proteins have been linked to microcephaly (MCPH), a neuro-developmental disease associated with small brain size. CPAP (MCPH6) and STIL (MCPH7) are required for centriole assembly, but it is unclear how mutations in them lead to microcephaly. We show that the TCP domain of CPAP constitutes a novel proline recognition domain that forms a 1:1 complex with a short, highly conserved target motif in STIL. Crystal structures of this complex reveal an unusual, all-β structure adopted by the TCP domain and explain how a microcephaly mutation in CPAP compromises complex formation. Through point mutations, we demonstrate that complex formation is essential for centriole duplication in vivo. Our studies provide the first structural insight into how the malfunction of centriole proteins results in human disease and also reveal that the CPAP–STIL interaction constitutes a conserved key step in centriole biogenesis. DOI: http://dx.doi.org/10.7554/eLife.01071.001 PMID:24052813

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

    NASA Astrophysics Data System (ADS)

    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

  15. Unexpected development of artistic talents.

    PubMed

    Gordon, N

    2005-12-01

    The development of exceptional and unexpected artistic skills at any age must be a matter of curiosity. This can occur among young children with severe learning difficulties, especially if they are autistic. Some examples of these so called idiot-savants are given, and the way in which their brains may function. It is also true that elderly people who suffer from frontotemporal dementia can find that they are able to express themselves in remarkable art forms. This can occur in other types of dementia, but then more often it is the changes that result in the paintings of established artists, for example in the paintings of de Kooning. Possible links between these two phenomenon are discussed, and it is suggested that in both instances it may be that if the brain is relieved of a number of functions it can concentrate on the remaining ones. Ways in which this may operate in both groups are reviewed. PMID:16344297

  16. Unexpected development of artistic talents

    PubMed Central

    Gordon, N

    2005-01-01

    The development of exceptional and unexpected artistic skills at any age must be a matter of curiosity. This can occur among young children with severe learning difficulties, especially if they are autistic. Some examples of these so called idiot-savants are given, and the way in which their brains may function. It is also true that elderly people who suffer from frontotemporal dementia can find that they are able to express themselves in remarkable art forms. This can occur in other types of dementia, but then more often it is the changes that result in the paintings of established artists, for example in the paintings of de Kooning. Possible links between these two phenomenon are discussed, and it is suggested that in both instances it may be that if the brain is relieved of a number of functions it can concentrate on the remaining ones. Ways in which this may operate in both groups are reviewed. PMID:16344297

  17. Cog5–Cog7 crystal structure reveals interactions essential for the function of a multisubunit tethering complex

    PubMed Central

    Ha, Jun Yong; Pokrovskaya, Irina D.; Climer, Leslie K.; Shimamura, Gregory R.; Kudlyk, Tetyana; Jeffrey, Philip D.; Lupashin, Vladimir V.; Hughson, Frederick M.

    2014-01-01

    The conserved oligomeric Golgi (COG) complex is required, along with SNARE and Sec1/Munc18 (SM) proteins, for vesicle docking and fusion at the Golgi. COG, like other multisubunit tethering complexes (MTCs), is thought to function as a scaffold and/or chaperone to direct the assembly of productive SNARE complexes at the sites of membrane fusion. Reflecting this essential role, mutations in the COG complex can cause congenital disorders of glycosylation. A deeper understanding of COG function and dysfunction will likely depend on elucidating its molecular structure. Despite some progress toward this goal, including EM studies of COG lobe A (subunits 1–4) and higher-resolution structures of portions of Cog2 and Cog4, the structures of COG’s eight subunits and the principles governing their assembly are mostly unknown. Here, we report the crystal structure of a complex between two lobe B subunits, Cog5 and Cog7. The structure reveals that Cog5 is a member of the complexes associated with tethering containing helical rods (CATCHR) fold family, with homology to subunits of other MTCs including the Dsl1, exocyst, and Golgi-associated retrograde protein (GARP) complexes. The Cog5–Cog7 interaction is analyzed in relation to the Dsl1 complex, the only other CATCHR-family MTC for which subunit interactions have been characterized in detail. Biochemical and functional studies validate the physiological relevance of the observed Cog5–Cog7 interface, indicate that it is conserved from yeast to humans, and demonstrate that its disruption in human cells causes defects in trafficking and glycosylation. PMID:25331899

  18. Diverse Roles and Interactions of the SWI/SNF Chromatin Remodeling Complex Revealed Using Global Approaches

    PubMed Central

    Davidov, Eugene; Gianoulis, Tara A.; Zhong, Guoneng; Rozowsky, Joel; Bhardwaj, Nitin; Gerstein, Mark B.; Snyder, Michael

    2011-01-01

    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 interactions

  19. Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry.

    PubMed

    Silva-Ayala, Daniela; López, Tomás; Gutiérrez, Michelle; Perrimon, Norbert; López, Susana; Arias, Carlos F

    2013-06-18

    Rotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the endosomal sorting complex required for transport (ESCRT) machinery were found. Here we provide evidence for a role of the ESCRT complex in the entry of simian and human RV strains in both monkey and human epithelial cells. In addition, the ESCRT-associated ATPase VPS4A and phospholipid lysobisphosphatidic acid, both crucial for the formation of intralumenal vesicles in multivesicular bodies, were also found to be required for cell entry. Interestingly, it seems that regardless of the molecules that rhesus RV and human RV strains use for cell-surface attachment and the distinct endocytic pathway used, all these viruses converge in early endosomes and use multivesicular bodies for cell entry. Furthermore, the small GTPases RHOA and CDC42, which regulate different types of clathrin-independent endocytosis, as well as early endosomal antigen 1 (EEA1), were found to be involved in this process. This work reports the direct involvement of the ESCRT machinery in the life cycle of a nonenveloped virus and highlights the complex mechanism that these viruses use to enter cells. It also illustrates the efficiency of high-throughput RNAi screenings as genetic tools for comprehensively studying the interaction between viruses and their host cells. PMID:23733942

  20. Characterization of Aquilegia Polycomb Repressive Complex 2 homologs reveals absence of imprinting.

    PubMed

    Gleason, Emily J; Kramer, Elena M

    2012-10-01

    Epigenetic regulation is important for maintaining gene expression patterns in multicellular organisms. The Polycomb Group (PcG) proteins form several complexes with important and deeply conserved epigenetic functions in both the plant and animal kingdoms. The plant Polycomb Repressive Complex 2 (PRC2) contains four core proteins, Enhancer of Zeste (E(z)), Suppressor of Zeste 12 (Su(z)12), Extra Sex Combs (ESC), and Multicopy Suppressor of IRA 1 (MSI1), and functions in many developmental transitions. In some plant species, including rice and Arabidopsis, duplications in the core PRC2 proteins allow the formation of PRC2s with distinct developmental functions. In addition, members of the plant specific VEL PHD family have been shown to associate with the PRC2 complex in Arabidopsis and may play a role in targeting the PRC2 to specific loci. Here we examine the evolution and expression of the PRC2 and VEL PHD families in Aquilegia, a member of the lower eudicot order Ranunculales and an emerging model for the investigation of plant ecology, evolution and developmental genetics. We find that Aquilegia has a relatively simple PRC2 with only one homolog of Su(z)12, ESC and MSI1 and two ancient copies of E(z), AqSWN and AqCLF. Aquilegia has four members of the VEL PHD family, three of which appear to be closely related to Arabidopsis proteins known to associate with the PRC2. The PRC2 and VEL PHD family proteins are expressed at a relatively constant level throughout Aquilegia vulgaris development, with the VEL PHD family and MSI1 expressed at higher levels during and after vernalization and in the inflorescence. Both AqSWN and AqCLF are expressed in Aquilegia endosperm but neither copy is imprinted. PMID:22796128

  1. Seismic Anisotropy Reveals a Large Magmatic Sill Complex below the Toba Caldera

    NASA Astrophysics Data System (ADS)

    Jaxybulatov, K.; Shapiro, N.; Koulakov, I.; Mordret, A.; Landes, M.; Sens-Schoenfelder, C.

    2014-12-01

    An understanding of the formation of large magmatic reservoirs is a key issue for the evaluation of possible strong volcanic eruptions in the future. Many geological observations of exposed past volcanic systems and geodynamic models have indicated that large magmatic reservoirs can build up over long periods of time, with small increments that promote vertical dykes and horizontally oriented sill intrusions. The typical size of such intrusions beneath presently active volcanoes is too small to be imaged with most geophysical methods. Here we show that large layered intrusion complexes at depth can be detected by measurements of the seismic anisotropy caused by the fine-scale layering. We have used data obtained from 42 stations between May and October 2008 on the Toba caldera complex (north Sumatra, Indonesia) to construct a 3D seismic model of the crust with using ambient noise tomography method. Approximately ~500 Rayleigh and Love waves were extracted from cross correlations of continuous records. Their group velocities were measured at periods between 5 and 19 s and, after a 2D regionalization, inverted into local 1D shear velocity profiles (VSH from Love and VSV from Rayleigh waves) using a Monte-Carlo method based on the Neighborhood Algorithm. All 1D profiles were combined into a final 3D model that shows a strong radial anisotropy below the Toba caldera at depths greater than 7 km. A plausible explanation of these observations is the presence of a large magmatic sill complex in the crust below 7 km in depth. Our data support the concept of the long-term incremental building up of magma bodies that leads to the largest volcanic eruptions.

  2. Tissue-specific regulatory circuits reveal variable modular perturbations across complex diseases.

    PubMed

    Marbach, Daniel; Lamparter, David; Quon, Gerald; Kellis, Manolis; Kutalik, Zoltán; Bergmann, Sven

    2016-04-01

    Mapping perturbed molecular circuits that underlie complex diseases remains a great challenge. We developed a comprehensive resource of 394 cell type- and tissue-specific gene regulatory networks for human, each specifying the genome-wide connectivity among transcription factors, enhancers, promoters and genes. Integration with 37 genome-wide association studies (GWASs) showed that disease-associated genetic variants-including variants that do not reach genome-wide significance-often perturb regulatory modules that are highly specific to disease-relevant cell types or tissues. Our resource opens the door to systematic analysis of regulatory programs across hundreds of human cell types and tissues (http://regulatorycircuits.org). PMID:26950747

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

    PubMed

    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. PMID:27441985

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

    PubMed Central

    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

  5. DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes

    PubMed Central

    Iftikhar, Romana; Ashfaq, Muhammad; Rasool, Akhtar; Hebert, Paul D. N.

    2016-01-01

    Although thrips are globally important crop pests and vectors of viral disease, species identifications are difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial COI-5ʹ (DNA barcode) region has proven effective for the identification of species in many groups of insect pests. We analyzed barcode sequence variation among 471 thrips from various plant hosts in north-central Pakistan. The Barcode Index Number (BIN) system assigned these sequences to 55 BINs, while the Automatic Barcode Gap Discovery detected 56 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 19% sequence divergence (range = 5.6% - 27%) at COI, while intraspecific distances averaged 0.6% (range = 0.0% - 7.6%). BIN analysis suggested that all intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Haplothrips reuteri, Thrips palmi, Thrips tabaci), and one predatory thrips (Aeolothrips intermedius) showed deep intraspecific divergences, providing evidence that each is a cryptic species complex. The study compiles the first barcode reference library for the thrips of Pakistan, and examines global haplotype diversity in four important pest thrips. PMID:26741134

  6. Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane.

    PubMed

    Löchte, Sara; Waichman, Sharon; Beutel, Oliver; You, Changjiang; Piehler, Jacob

    2014-11-10

    Interactions of proteins in the plasma membrane are notoriously challenging to study under physiological conditions. We report in this paper a generic approach for spatial organization of plasma membrane proteins into micropatterns as a tool for visualizing and quantifying interactions with extracellular, intracellular, and transmembrane proteins in live cells. Based on a protein-repellent poly(ethylene glycol) polymer brush, micropatterned surface functionalization with the HaloTag ligand for capturing HaloTag fusion proteins and RGD peptides promoting cell adhesion was devised. Efficient micropatterning of the type I interferon (IFN) receptor subunit IFNAR2 fused to the HaloTag was achieved, and highly specific IFN binding to the receptor was detected. The dynamics of this interaction could be quantified on the single molecule level, and IFN-induced receptor dimerization in micropatterns could be monitored. Assembly of active signaling complexes was confirmed by immunostaining of phosphorylated Janus family kinases, and the interaction dynamics of cytosolic effector proteins recruited to the receptor complex were unambiguously quantified by fluorescence recovery after photobleaching. PMID:25385185

  7. Eigencentrality based on dissimilarity measures reveals central nodes in complex networks

    NASA Astrophysics Data System (ADS)

    Alvarez-Socorro, A. J.; Herrera-Almarza, G. C.; González-Díaz, L. A.

    2015-11-01

    One of the most important problems in complex network’s theory is the location of the entities that are essential or have a main role within the network. For this purpose, the use of dissimilarity measures (specific to theory of classification and data mining) to enrich the centrality measures in complex networks is proposed. The centrality method used is the eigencentrality which is based on the heuristic that the centrality of a node depends on how central are the nodes in the immediate neighbourhood (like rich get richer phenomenon). This can be described by an eigenvalues problem, however the information of the neighbourhood and the connections between neighbours is not taken in account, neglecting their relevance when is one evaluates the centrality/importance/influence of a node. The contribution calculated by the dissimilarity measure is parameter independent, making the proposed method is also parameter independent. Finally, we perform a comparative study of our method versus other methods reported in the literature, obtaining more accurate and less expensive computational results in most cases.

  8. Super-resolution microscopy reveals LINC complex recruitment at nuclear indentation sites.

    PubMed

    Versaevel, Marie; Braquenier, Jean-Baptiste; Riaz, Maryam; Grevesse, Thomas; Lantoine, Joséphine; Gabriele, Sylvain

    2014-01-01

    Increasing evidences show that the actin cytoskeleton is a key parameter of the nuclear remodeling process in response to the modifications of cellular morphology. However, detailed information on the interaction between the actin cytoskeleton and the nuclear lamina was still lacking. We addressed this question by constraining endothelial cells on rectangular fibronectin-coated micropatterns and then using Structured Illumination Microscopy (SIM) to observe the interactions between actin stress fibers, nuclear lamina and LINC complexes at a super-resolution scale. Our results show that tension in apical actin stress fibers leads to deep nuclear indentations that significantly deform the nuclear lamina. Interestingly, indented nuclear zones are characterized by a local enrichment of LINC complexes, which anchor apical actin fibers to the nuclear lamina. Moreover, our findings indicate that nuclear indentations induce the formation of segregated domains of condensed chromatin. However, nuclear indentations and condensed chromatin domains are not irreversible processes and both can relax in absence of tension in apical actin stress fibers. PMID:25482017

  9. DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes.

    PubMed

    Iftikhar, Romana; Ashfaq, Muhammad; Rasool, Akhtar; Hebert, Paul D N

    2016-01-01

    Although thrips are globally important crop pests and vectors of viral disease, species identifications are difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial COI-5' (DNA barcode) region has proven effective for the identification of species in many groups of insect pests. We analyzed barcode sequence variation among 471 thrips from various plant hosts in north-central Pakistan. The Barcode Index Number (BIN) system assigned these sequences to 55 BINs, while the Automatic Barcode Gap Discovery detected 56 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 19% sequence divergence (range = 5.6% - 27%) at COI, while intraspecific distances averaged 0.6% (range = 0.0% - 7.6%). BIN analysis suggested that all intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Haplothrips reuteri, Thrips palmi, Thrips tabaci), and one predatory thrips (Aeolothrips intermedius) showed deep intraspecific divergences, providing evidence that each is a cryptic species complex. The study compiles the first barcode reference library for the thrips of Pakistan, and examines global haplotype diversity in four important pest thrips. PMID:26741134

  10. Polycomb repressive complex 2 structure with inhibitor reveals a mechanism of activation and drug resistance

    PubMed Central

    Brooun, Alexei; Gajiwala, Ketan S.; Deng, Ya-Li; Liu, Wei; Bolaños, Ben; Bingham, Patrick; He, You-Ai; Diehl, Wade; Grable, Nicole; Kung, Pei-Pei; Sutton, Scott; Maegley, Karen A.; Yu, Xiu; Stewart, Al E.

    2016-01-01

    Polycomb repressive complex 2 (PRC2) mediates gene silencing through chromatin reorganization by methylation of histone H3 lysine 27 (H3K27). Overexpression of the complex and point mutations in the individual subunits of PRC2 have been shown to contribute to tumorigenesis. Several inhibitors of the PRC2 activity have shown efficacy in EZH2-mutated lymphomas and are currently in clinical development, although the molecular basis of inhibitor recognition remains unknown. Here we report the crystal structures of the inhibitor-bound wild-type and Y641N PRC2. The structures illuminate an important role played by a stretch of 17 residues in the N-terminal region of EZH2, we call the activation loop, in the stimulation of the enzyme activity, inhibitor recognition and the potential development of the mutation-mediated drug resistance. The work presented here provides new avenues for the design and development of next-generation PRC2 inhibitors through establishment of a structure-based drug design platform. PMID:27122193

  11. Multivariate and Multiscale Dependence in the Global Climate System Revealed Through Complex Networks

    SciTech Connect

    Steinhaeuser, Karsten J K; Ganguly, Auroop R; Chawla, Nitesh

    2011-01-01

    A systematic characterization of multivariate dependence at multiple spatio-temporal scales is critical to understanding climate system dynamics and improving predictive ability from models and data. However, dependence structures in climate are complex due to nonlinear dynamical generating processes, long-range spatial and long-memory temporal relationships, as well as low-frequency variability. Here we utilize complex networks to explore dependence in climate data. Specifically, networks constructed from reanalysis-based atmospheric variables over oceans and partitioned with community detection methods demonstrate the potential to capture regional and global dependence structures within and among climate variables. Proximity-based dependence as well as long-range spatial relationships are examined along with their evolution over time, yielding new insights on ocean meteorology. The tools are implicitly validated by confirming conceptual understanding about aggregate correlations and teleconnections. Our results also suggest a close similarity of observed dependence patterns in relative humidity and horizontal wind speed over oceans. In addition, updraft velocity, which relates to convective activity over the oceans, exhibits short spatiotemporal decorrelation scales but long-range dependence over time. The multivariate and multi-scale dependence patterns broadly persist over multiple time windows. Our findings motivate further investigations of dependence structures among observations, reanalysis and model-simulated data to enhance process understanding, assess model reliability and improve regional climate predictions.

  12. Complex mode of inheritance in holoprosencephaly revealed by whole exome sequencing.

    PubMed

    Mouden, C; Dubourg, C; Carré, W; Rose, S; Quelin, C; Akloul, L; Hamdi-Rozé, H; Viot, G; Salhi, H; Darnault, P; Odent, S; Dupé, V; David, V

    2016-06-01

    Holoprosencephaly (HPE) is the most common congenital cerebral malformation, characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been associated with HPE and are often inherited from an unaffected parent, underlying complex genetic bases. It is now emerging that HPE may result from a combination of multiple genetic events, rather than from a single heterozygous mutation. To explore this hypothesis, we undertook whole exome sequencing and targeted high-throughput sequencing approaches to identify mutations in HPE subjects. Here, we report two HPE families in which two mutations are implicated in the disease. In the first family presenting two foetuses with alobar and semi-lobar HPE, we found mutations in two genes involved in HPE, SHH and DISP1, inherited respectively from the father and the mother. The second reported case is a family with a 9-year-old girl presenting lobar HPE, harbouring two compound heterozygous mutations in DISP1. Together, these cases of digenic inheritance and autosomal recessive HPE suggest that in some families, several genetic events are necessary to cause HPE. This study highlights the complexity of HPE inheritance and has to be taken into account by clinicians to improve HPE genetic counselling. PMID:26748417

  13. Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations

    PubMed Central

    Weninger, Keith; Bowen, Mark E.; Chu, Steven; Brunger, Axel T.

    2003-01-01

    Vesicle fusion in eukaryotes is thought to involve the assembly of a highly conserved family of proteins termed soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a highly stable parallel four-helix bundle. We have used intermolecular single-molecule fluorescence resonance energy transfer to characterize preassembled neuronal SNARE complexes consisting of syntaxin, synaptobrevin, and synaptosome-associated protein of 25 kDa on deposited lipid bilayers. Surprisingly, we found a mixture of parallel as well as antiparallel configurations involving the SNARE motifs of syntaxin and synaptobrevin as well as those of syntaxin and synaptosome-associated protein of 25 kDa. The subpopulation with the parallel four-helix bundle configuration could be greatly enriched by an additional purification step in the presence of denaturant, indicating that the parallel configuration is the energetically most favorable state. Interconversion between the configurations was not observed. From this observation, we infer the conversion rate to be <1.5 h–1. The existence of antiparallel configurations suggests a regulatory role of chaperones, such as N-ethylmaleimide-sensitive factor, or the membrane environment during SNARE complex assembly in vivo, and it could be a partial explanation for the relatively slow rates of vesicle fusion observed by reconstituted fusion experiments in vitro. PMID:14657376

  14. Adaptive and selective seed abortion reveals complex conditional decision making in plants.

    PubMed

    Meyer, Katrin M; Soldaat, Leo L; Auge, Harald; Thulke, Hans-Hermann

    2014-03-01

    Behavior is traditionally attributed to animals only. Recently, evidence for plant behavior is accumulating, mostly from plant physiological studies. Here, we provide ecological evidence for complex plant behavior in the form of seed abortion decisions conditional on internal and external cues. We analyzed seed abortion patterns of barberry plants exposed to seed parasitism and different environmental conditions. Without abortion, parasite infestation of seeds can lead to loss of all seeds in a fruit. We statistically tested a series of null models with Monte Carlo simulations to establish selectivity and adaptiveness of the observed seed abortion patterns. Seed abortion was more frequent in parasitized fruits and fruits from dry habitats. Surprisingly, seed abortion occurred with significantly greater probability if there was a second intact seed in the fruit. This strategy provides a fitness benefit if abortion can prevent a sibling seed from coinfestation and if nonabortion of an infested but surviving single seed saves resources invested in the fruit coat. Ecological evidence for complex decision making in plants thus includes a structural memory (the second seed), simple reasoning (integration of inner and outer conditions), conditional behavior (abortion), and anticipation of future risks (seed predation). PMID:24561600

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

    PubMed

    Derré, Isabelle; Pypaert, Marc; Dautry-Varsat, Alice; Agaisse, Hervé

    2007-10-26

    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. PMID:17967059

  16. Structure of a murine norovirus NS6 protease-product complex revealed by adventitious crystallisation.

    PubMed

    Leen, Eoin N; Baeza, Gabriela; Curry, Stephen

    2012-01-01

    Murine noroviruses have emerged as a valuable tool for investigating the molecular basis of infection and pathogenesis of the closely related human noroviruses, which are the major cause of non-bacterial gastroenteritis. The replication of noroviruses relies on the proteolytic processing of a large polyprotein precursor into six non-structural proteins (NS1-2, NS3, NS4, NS5, NS6(pro), NS7(pol)) by the virally-encoded NS6 protease. We report here the crystal structure of MNV NS6(pro), which has been determined to a resolution of 1.6 Å. Adventitiously, the crystal contacts are mediated in part by the binding of the C-terminus of NS6(pro) within the peptide-binding cleft of a neighbouring molecule. This insertion occurs for both molecules in the asymmetric unit of the crystal in a manner that is consistent with physiologically-relevant binding, thereby providing two independent views of a protease-peptide complex. Since the NS6(pro) C-terminus is formed in vivo by NS6(pro) processing, these crystal contacts replicate the protease-product complex that is formed immediately following cleavage of the peptide bond at the NS6-NS7 junction. The observed mode of binding of the C-terminal product peptide yields new insights into the structural basis of NS6(pro) specificity. PMID:22685603

  17. Eigencentrality based on dissimilarity measures reveals central nodes in complex networks

    PubMed Central

    Alvarez-Socorro, A. J.; Herrera-Almarza, G. C.; González-Díaz, L. A.

    2015-01-01

    One of the most important problems in complex network’s theory is the location of the entities that are essential or have a main role within the network. For this purpose, the use of dissimilarity measures (specific to theory of classification and data mining) to enrich the centrality measures in complex networks is proposed. The centrality method used is the eigencentrality which is based on the heuristic that the centrality of a node depends on how central are the nodes in the immediate neighbourhood (like rich get richer phenomenon). This can be described by an eigenvalues problem, however the information of the neighbourhood and the connections between neighbours is not taken in account, neglecting their relevance when is one evaluates the centrality/importance/influence of a node. The contribution calculated by the dissimilarity measure is parameter independent, making the proposed method is also parameter independent. Finally, we perform a comparative study of our method versus other methods reported in the literature, obtaining more accurate and less expensive computational results in most cases. PMID:26603652

  18. Single muscle fiber proteomics reveals unexpected mitochondrial specialization

    PubMed Central

    Murgia, Marta; Nagaraj, Nagarjuna; Deshmukh, Atul S; Zeiler, Marlis; Cancellara, Pasqua; Moretti, Irene; Reggiani, Carlo; Schiaffino, Stefano; Mann, Matthias

    2015-01-01

    Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high-sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity. PMID:25643707

  19. Water in stars: expected and unexpected

    NASA Astrophysics Data System (ADS)

    Tsuji, T.; Aoki, W.; Ohnaka, K.

    1999-03-01

    We have confirmed the presence of water in the early M giant α Cet (M1.5III) and supergiant KK Per (M2Iab) by the highest resolution grating mode of SWS, but this result is quite unexpected from present model atmospheres. In late M giant and supergiant stars, water observed originates partly in the photosphere as expected by the model atmospheres, but ISO SWS has revealed that the 2.7 mic\\ absorption bands appear to be somewhat stronger than predicted while 6.5 mic\\ bands weaker, indicating the contamination by an emission component. In the mid-infrared region extending to 45 mic, pure rotation lines of hho\\ appear as distinct emission on the high resolution SWS spectra of 30g Her (M7III) and S Per (M4-7Ia), along with the dust emission at 10, 13, 20 mic\\ and a new unidentified feature at 30 mic. Thus, together with the dust, water contributes to the thermal balance of the outer atmosphere already in the mid-infrared. The excitation temperature of hho\\ gas is estimated to be 500 - 1000 K. In view of this result for late M (super)giants, unexpected water observed in early M (super)giants should also be of non-photospheric in origin. Thus, ISO has finally established the presence of a new component of the outer atmosphere - a warm molecular envelope - in red giant and supergiant stars from early to late types. Such a rather warm molecular envelope will be a site of various activities such as chemical reactions, dust formation, mass-outflow etc.

  20. Dynamics of ribosome scanning and recycling revealed by translation complex profiling.

    PubMed

    Archer, Stuart K; Shirokikh, Nikolay E; Beilharz, Traude H; Preiss, Thomas

    2016-07-28

    Regulation of messenger RNA translation is central to eukaryotic gene expression control. Regulatory inputs are specified by them RNA untranslated regions (UTRs) and often target translation initiation. Initiation involves binding of the 40S ribosomal small subunit (SSU) and associated eukaryotic initiation factors (eIFs)near the mRNA 5′ cap; the SSU then scans in the 3′ direction until it detects the start codon and is joined by the 60S ribosomal large subunit (LSU) to form the 80S ribosome. Scanning and other dynamic aspects of the initiation model have remained as conjectures because methods to trap early intermediates were lacking. Here we uncover the dynamics of the complete translation cycle in live yeast cells using translation complex profile sequencing (TCP-seq), a method developed from the ribosome profiling approach. We document scanning by observing SSU footprints along 5′ UTRs. Scanning SSU have 5′-extended footprints (up to~75 nucleotides), indicative of additional interactions with mRNA emerging from the exit channel, promoting forward movement. We visualized changes in initiation complex conformation as SSU footprints coalesced into three major sizes at start codons (19, 29 and 37 nucleotides). These share the same 5′ start site but differ at the 3′ end, reflecting successive changes at the entry channel from an open to a closed state following start codon recognition. We also observe SSU 'lingering' at stop codons after LSU departure. Our results underpin mechanistic models of translation initiation and termination, built on decades of biochemical and structural investigation, with direct genome-wide in vivo evidence. Our approach captures ribosomal complexes at all phases of translation and will aid in studying translation dynamics in diverse cellular contexts. Dysregulation of translation is common in disease and, for example, SSU scanning is a target of anti-cancer drug development. TCP-seq will prove useful in discerning differences

  1. Chemical Complexity in the Shocked Outflow L1157 Revealed by CARMA

    NASA Astrophysics Data System (ADS)

    Dollhopf, Niklaus M.; McGuire, Brett A.; Carroll, P. Brandon; Remijan, Anthony J.

    2015-01-01

    Amino acids, the complex organic molecules which are the building blocks of life, have been found in meteoritic samples and, most recently, in samples from Comet Wild-2. Yet, no amino acids have been detected in the gas-phase in the interstellar medium, which seeds and enriches these meteorites and comets. Glycine, the simplest amino acid, has been shown to form in the laboratory through the reaction of hydroxylamine (NH2OH) with acetic acid (CH3COOH), a known interstellar molecule. This has prompted a move to search for NH2OH as a proxy of identifying regions where subsequent searches for glycine may prove the most fruitful.A search for NH2OH was conducted in seven diverse, molecule-rich sources and resulted in non-detections for all seven (Pulliam, et al. 2012). Theoretical work suggested the temperature of the sources was perhaps too low for NH2OH to thermally-desorb into the gas phase. Searches in shocked molecular regions, however, may overcome this barrier, as complex molecules are non-thermally liberated into the gas-phase by these shocks.Here, we present results from a targeted search toward the prototypical shocked outflow L1157. L1157-B0, -B1, and -B2 are shocked regions within the outflow from the infrared source L1157-mm. Using observations from the Combined Array for Research in Millimeter-wave Astronomy (CARMA), we have mapped a variety of molecular tracers in the region and conducted an interferometric search for NH2OH with typical spatial resolutions of ~3'. We find that the prototypical complex molecule methanol (CH3OH) peaks in B2, the newer shock. We compare this with the distributions of HCN and HCO+ and discuss the implications for chemical evolution within the region. HCN, used as a density tracer, also peaks in B2 while HCO+ is shown as diffuse throughout B0. We also present the first maps of isocyanic acid (HNCO) in L1157. HNCO is found to peak in B2, cospatial with CH3OH and HCN. Finally, we report a non-detection of three NH2OH transitions

  2. Ancient mitochondrial DNA analysis reveals complexity of indigenous North American turkey domestication.

    PubMed

    Speller, Camilla F; Kemp, Brian M; Wyatt, Scott D; Monroe, Cara; Lipe, William D; Arndt, Ursula M; Yang, Dongya Y

    2010-02-16

    Although the cultural and nutritive importance of the turkey (Meleagris gallopavo) to precontact Native Americans and contemporary people worldwide is clear, little is known about the domestication of this bird compared to other domesticates. Mitochondrial DNA analysis of 149 turkey bones and 29 coprolites from 38 archaeological sites (200 BC-AD 1800) reveals a unique domesticated breed in the precontact Southwestern United States. Phylogeographic analyses indicate that this domestic breed originated from outside the region, but rules out the South Mexican domestic turkey (Meleagris gallopavo gallopavo) as a progenitor. A strong genetic bottleneck within the Southwest turkeys also reflects intensive human selection and breeding. This study points to at least two occurrences of turkey domestication in precontact North America and illuminates the intensity and sophistication of New World animal breeding practices. PMID:20133614

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

    SciTech Connect

    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

    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.

  4. [Complex metabolic disorders revealing a gastric ulcer of the bulb. A case report].

    PubMed

    Neffati, F; Hellara, I; Jelizi, M A; Bahri, J; Douki, W; Amor, A Ben; Najjar, M F

    2009-01-01

    We report the case of a 54-year-old man, without particular pathological antecedents admitted to the emergency of the university hospital of Monastir, for right renal colic. Radiography of the urinary tract without preparation and renal echography showed bilateral renal lithiasis and a right ureteral lithiasis. The interrogation revealed concept of vomiting after which the patient felt relieved. The biological assessment objectified an hypochloremic metabolic alcalosis, an increase in the anion gap, a severe impaired renal function of obstructive origin and an hypokaliemia. The presence of the lithiasis did not explain on its own the metabolic disorders of this patient. The other investigations showed that initial pathology was an evolutionary bulb ulcer into pre-stenosis justifying treatment by omeprazole and explaining the biological disorders. PMID:19654086

  5. Ancient mitochondrial DNA analysis reveals complexity of indigenous North American turkey domestication

    PubMed Central

    Speller, Camilla F.; Kemp, Brian M.; Wyatt, Scott D.; Monroe, Cara; Lipe, William D.; Arndt, Ursula M.; Yang, Dongya Y.

    2010-01-01

    Although the cultural and nutritive importance of the turkey (Meleagris gallopavo) to precontact Native Americans and contemporary people worldwide is clear, little is known about the domestication of this bird compared to other domesticates. Mitochondrial DNA analysis of 149 turkey bones and 29 coprolites from 38 archaeological sites (200 BC–AD 1800) reveals a unique domesticated breed in the precontact Southwestern United States. Phylogeographic analyses indicate that this domestic breed originated from outside the region, but rules out the South Mexican domestic turkey (Meleagris gallopavo gallopavo) as a progenitor. A strong genetic bottleneck within the Southwest turkeys also reflects intensive human selection and breeding. This study points to at least two occurrences of turkey domestication in precontact North America and illuminates the intensity and sophistication of New World animal breeding practices. PMID:20133614

  6. Protein Configuration Landscape Fluctuations Revealed by Exciton Transition Polarizations in Single Light Harvesting Complexes.

    PubMed

    Tubasum, Sumera; Torbjörnsson, Magne; Yadav, Dheerendra; Camacho, Rafael; Söderlind, Gustaf; Scheblykin, Ivan G; Pullerits, Tõnu

    2016-02-01

    Protein is a flexible material with broad distribution of conformations forming an energy landscape of quasi-stationary states. Disentangling the system dynamics along this landscape is the key for understanding the functioning of the protein. Here we studied a photosynthetic antenna pigment-protein complex LH2 with single molecule two-dimensional polarization imaging. Modeling based on the Redfield relaxation theory well describes the observed polarization properties of LH2 fluorescence and fluorescence excitation, strongly suggesting that at 77 K the conformational subspace of the LH2 is limited to about three configurations with relatively frequent switching among each other. At room temperature the next level of fluctuations determines the conformational dynamics. The results support the multitier model of the energy landscape of proteins and demonstrate the potential of the method for the studies of structural dynamics in proteins. PMID:26741912

  7. Two Allergen Model Reveals Complex Relationship Between IgE Cross-Linking and Degranulation

    PubMed Central

    Handlogten, Michael W.; Deak, Peter E.; Bilgicer, Basar

    2014-01-01

    Summary Allergy is an immune response to complex mixtures of multiple allergens yet current models use a single synthetic allergen. Multiple allergens were modeled using two well-defined tetravalent allergens each specific for a distinct IgE thus enabling a systematic approach to evaluate the effect of each allergen and percent of allergen specific IgE on mast cell degranulation. We found the overall degranulation response caused by two allergens is additive for low allergen concentrations or low percent specific IgE, does not change for moderate allergen concentrations with moderate to high percent specific IgE, and is reduced for high allergen concentrations with moderate to high percent specific IgE. These results provide further evidence that supra-optimal IgE cross-linking decreases the degranulation response and establishes the two allergen model as a relevant experimental system to elucidate mast cell degranulation mechanisms. PMID:25308278

  8. Potential of metabolomics to reveal Burkholderia cepacia complex pathogenesis and antibiotic resistance

    PubMed Central

    Shommu, Nusrat S.; Vogel, Hans J.; Storey, Douglas G.

    2015-01-01

    The Burkholderia cepacia complex (Bcc) is a collection of closely related, genetically distinct, ecologically diverse species known to cause life-threatening infections in cystic fibrosis (CF) patients. By virtue of a flexible genomic structure and diverse metabolic activity, Bcc bacteria employ a wide array of virulence factors for pathogenesis in CF patients and have developed resistance to most of the commonly used antibiotics. However, the mechanism of pathogenesis and antibiotic resistance is still not fully understood. This mini review discusses the established and potential virulence determinants of Bcc and some of the contemporary strategies including transcriptomics and proteomics used to identify these traits. We also propose the application of metabolic profiling, a cost-effective modern-day approach to achieve new insights. PMID:26217312

  9. Antidiabetic phospholipid-nuclear receptor complex reveals the mechanism for phospholipid-driven gene regulation

    SciTech Connect

    Musille, Paul M; Pathak, Manish C; Lauer, Janelle L; Hudson, William H; Griffin, Patrick R; Ortlund, Eric A

    2013-01-31

    The human nuclear receptor liver receptor homolog-1 (LRH-1) has an important role in controlling lipid and cholesterol homeostasis and is a potential target for the treatment of diabetes and hepatic diseases. LRH-1 is known to bind phospholipids, but the role of phospholipids in controlling LRH-1 activation remains highly debated. Here we describe the structure of both apo LRH-1 and LRH-1 in complex with the antidiabetic phospholipid dilauroylphosphatidylcholine (DLPC). Together with hydrogen-deuterium exchange MS and functional data, our studies show that DLPC binding is a dynamic process that alters co-regulator selectivity. We show that the lipid-free receptor undergoes previously unrecognized structural fluctuations, allowing it to interact with widely expressed co-repressors. These observations enhance our understanding of LRH-1 regulation and highlight its importance as a new therapeutic target for controlling diabetes.

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

    PubMed

    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. PMID:24291091

  11. Shadows of complexity: what biological networks reveal about epistasis and pleiotropy.

    PubMed

    Tyler, Anna L; Asselbergs, Folkert W; Williams, Scott M; Moore, Jason H

    2009-02-01

    Pleiotropy, in which one mutation causes multiple phenotypes, has traditionally been seen as a deviation from the conventional observation in which one gene affects one phenotype. Epistasis, or gene-gene interaction, has also been treated as an exception to the Mendelian one gene-one phenotype paradigm. This simplified perspective belies the pervasive complexity of biology and hinders progress toward a deeper understanding of biological systems. We assert that epistasis and pleiotropy are not isolated occurrences, but ubiquitous and inherent properties of biomolecular networks. These phenomena should not be treated as exceptions, but rather as fundamental components of genetic analyses. A systems level understanding of epistasis and pleiotropy is, therefore, critical to furthering our understanding of human genetics and its contribution to common human disease. Finally, graph theory offers an intuitive and powerful set of tools with which to study the network bases of these important genetic phenomena. PMID:19204994

  12. Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy

    PubMed Central

    Masedunskas, Andrius; Sramkova, Monika; Parente, Laura; Sales, Katiuchia Uzzun; Amornphimoltham, Panomwat; Bugge, Thomas H.; Weigert, Roberto

    2011-01-01

    The regulation and the dynamics of membrane trafficking events have been studied primarily in in vitro models that often do not fully reflect the functional complexity found in a living multicellular organism. Here we used intravital microscopy in the salivary glands of live rodents to investigate regulated exocytosis, a fundamental process in all of the secretory organs. We found that β-adrenergic stimulation elicits exocytosis of large secretory granules, which gradually collapse with the apical plasma membrane without any evidence of compound exocytosis, as was previously described. Furthermore, we show that the driving force required to complete the collapse of the granules is provided by the recruitment of F-actin and nonmuscle myosin II on the granule membranes that is triggered upon fusion with the plasma membrane. Our results provide information on the machinery controlling regulated secretion and show that intravital microscopy provides unique opportunities to address fundamental questions in cell biology under physiological conditions. PMID:21808006

  13. Dengue in China: Comprehensive Phylogenetic Evaluation Reveals Evidence of Endemicity and Complex Genetic Diversity.

    PubMed

    Chen, Rubing; Han, Guan-Zhu

    2016-01-01

    Despite the increasing threat of dengue outbreaks in China, it is still considered as an imported disease and its introduction and/or circulation patterns remain obscure. On the basis of the most extensive phylogenetic analysis to date, we showed highly complex genetic diversity of dengue viruses (DENVs) in south China with up to 20 different clades/lineages from multiple serotypes co-circulating in the same year. Despite that most of these clades/lineages were resulted from imported cases, evidence of local persistence of DENV serotype 1 (DENV-1) was observed, indicating its potential endemicity in Guangdong province. This study, therefore, provided an overview of DENV genetic diversity and evolutionary dynamics in China, which will be useful for developing policies to prevent and control future dengue outbreaks in China. PMID:26458780

  14. Analysis of the Mitochondrial Genome in Hypomyces aurantius Reveals a Novel Twintron Complex in Fungi.

    PubMed

    Deng, Youjin; Zhang, Qihui; Ming, Ray; Lin, Longji; Lin, Xiangzhi; Lin, Yiying; Li, Xiao; Xie, Baogui; Wen, Zhiqiang

    2016-01-01

    Hypomyces aurantius is a mycoparasite that causes cobweb disease, a most serious disease of cultivated mushrooms. Intra-species identification is vital for disease control, however the lack of genomic data makes development of molecular markers challenging. Small size, high copy number, and high mutation rate of fungal mitochondrial genome makes it a good candidate for intra and inter species differentiation. In this study, the mitochondrial genome of H. H.a0001 was determined from genomic DNA using Illumina sequencing. The roughly 72 kb genome shows all major features found in other Hypocreales: 14 common protein genes, large and small subunit rRNAs genes and 27 tRNAs genes. Gene arrangement comparison showed conserved gene orders in Hypocreales mitochondria are relatively conserved, with the exception of Acremonium chrysogenum and Acremonium implicatum. Mitochondrial genome comparison also revealed that intron length primarily contributes to mitogenome size variation. Seventeen introns were detected in six conserved genes: five in cox1, four in rnl, three in cob, two each in atp6 and cox3, and one in cox2. Four introns were found to contain two introns or open reading frames: cox3-i2 is a twintron containing two group IA type introns; cox2-i1 is a group IB intron encoding two homing endonucleases; and cox1-i4 and cox1-i3 both contain two open reading frame (ORFs). Analyses combining secondary intronic structures, insertion sites, and similarities of homing endonuclease genes reveal two group IA introns arranged side by side within cox3-i2. Mitochondrial data for H. aurantius provides the basis for further studies relating to population genetics and species identification. PMID:27376282

  15. mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus.

    PubMed

    Legendre, Matthieu; Audic, Stéphane; Poirot, Olivier; Hingamp, Pascal; Seltzer, Virginie; Byrne, Deborah; Lartigue, Audrey; Lescot, Magali; Bernadac, Alain; Poulain, Julie; Abergel, Chantal; Claverie, Jean-Michel

    2010-05-01

    Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic "virion factory," we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5' and 3' extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus "virophage." These results-validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)--will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system. PMID:20360389

  16. Analysis of the Mitochondrial Genome in Hypomyces aurantius Reveals a Novel Twintron Complex in Fungi

    PubMed Central

    Deng, Youjin; Zhang, Qihui; Ming, Ray; Lin, Longji; Lin, Xiangzhi; Lin, Yiying; Li, Xiao; Xie, Baogui; Wen, Zhiqiang

    2016-01-01

    Hypomyces aurantius is a mycoparasite that causes cobweb disease, a most serious disease of cultivated mushrooms. Intra-species identification is vital for disease control, however the lack of genomic data makes development of molecular markers challenging. Small size, high copy number, and high mutation rate of fungal mitochondrial genome makes it a good candidate for intra and inter species differentiation. In this study, the mitochondrial genome of H. H.a0001 was determined from genomic DNA using Illumina sequencing. The roughly 72 kb genome shows all major features found in other Hypocreales: 14 common protein genes, large and small subunit rRNAs genes and 27 tRNAs genes. Gene arrangement comparison showed conserved gene orders in Hypocreales mitochondria are relatively conserved, with the exception of Acremonium chrysogenum and Acremonium implicatum. Mitochondrial genome comparison also revealed that intron length primarily contributes to mitogenome size variation. Seventeen introns were detected in six conserved genes: five in cox1, four in rnl, three in cob, two each in atp6 and cox3, and one in cox2. Four introns were found to contain two introns or open reading frames: cox3-i2 is a twintron containing two group IA type introns; cox2-i1 is a group IB intron encoding two homing endonucleases; and cox1-i4 and cox1-i3 both contain two open reading frame (ORFs). Analyses combining secondary intronic structures, insertion sites, and similarities of homing endonuclease genes reveal two group IA introns arranged side by side within cox3-i2. Mitochondrial data for H. aurantius provides the basis for further studies relating to population genetics and species identification. PMID:27376282

  17. Ribonomic analysis of human DZIP1 reveals its involvement in ribonucleoprotein complexes and stress granules

    PubMed Central

    2014-01-01

    Background DZIP1 (DAZ-interacting protein 1) has been described as a component of the Hh signaling pathway with a putative regulatory role in ciliogenesis. DZIP1 interacts with DAZ RNA binding proteins in embryonic stem cells and human germ cells suggesting a role in mRNA regulation. Results We investigated DZIP1 function in HeLa cells and its involvement in ribonucleoprotein complexes. DZIP1 was predominantly located in granules in the cytoplasm. Under oxidative stress conditions, DZIP1 re-localized to stress granules. DZIP appears to be important for the formation of stress granules during the stress response. We used immunoprecipitation assays with antibodies against DZIP1 and microarray hybridization to identify mRNAs associated with DZIP1. The genetic networks formed by the DZIP1-associated mRNAs were involved in cell cycle and gene expression regulation. DZIP1 is involved in the Hedgehog signaling pathway. We used cyclopamine, a specific inhibitor of this pathway, to analyze the expression of DZIP1 and its associated mRNAs. The abundance of DZIP1-associated mRNAs increased with treatment; however, the silencing or overexpression of DZIP1 in HeLa cells had no effect on the accumulation of the associated mRNAs. Polysomal profile analysis by sucrose gradient centrifugation demonstrated the presence of DZIP1 in the polysomal fraction. Conclusions Our results suggest that DZIP1 is part of an RNP complex that occupies various subcellular locations. The diversity of the mRNAs associated with DZIP1 suggests that this protein is a component of different RNPs associated with translating polysomes and with RNA granules. PMID:24993635

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

    PubMed

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

    2014-09-01

    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

  19. Copper Complexation Screen Reveals Compounds with Potent Antibiotic Properties against Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Haeili, Mehri; Moore, Casey; Davis, Christopher J. C.; Cochran, James B.; Shah, Santosh; Shrestha, Tej B.; Zhang, Yaofang; Bossmann, Stefan H.; Benjamin, William H.

    2014-01-01

    Macrophages take advantage of the antibacterial properties of copper ions in the killing of bacterial intruders. However, despite the importance of copper for innate immune functions, coordinated efforts to exploit copper ions for therapeutic interventions against bacterial infections are not yet in place. Here we report a novel high-throughput screening platform specifically developed for the discovery and characterization of compounds with copper-dependent antibacterial properties toward methicillin-resistant Staphylococcus aureus (MRSA). We detail how one of the identified compounds, glyoxal-bis(N4-methylthiosemicarbazone) (GTSM), exerts its potent strictly copper-dependent antibacterial properties on MRSA. Our data indicate that the activity of the GTSM-copper complex goes beyond the general antibacterial effects of accumulated copper ions and suggest that, in contrast to prevailing opinion, copper complexes can indeed exhibit species- and target-specific activities. Based on experimental evidence, we propose that copper ions impose structural changes upon binding to the otherwise inactive GTSM ligand and transfer antibacterial properties to the chelate. In turn, GTSM determines target specificity and utilizes a redox-sensitive release mechanism through which copper ions are deployed at or in close proximity to a putative target. According to our proof-of-concept screen, copper activation is not a rare event and even extends to already established drugs. Thus, copper-activated compounds could define a novel class of anti-MRSA agents that amplify copper-dependent innate immune functions of the host. To this end, we provide a blueprint for a high-throughput drug screening campaign which considers the antibacterial properties of copper ions at the host-pathogen interface. PMID:24752262

  20. Collaborative play in young children as a complex dynamic system: revealing gender related differences.

    PubMed

    Steenbeek, Henderien; van der Aalsvoort, Diny; van Geert, Paul

    2014-07-01

    This study was focused on the role of gender-related differences in collaborative play, by examining properties of play as a complex system, and by using micro-genetic analysis techniques. A complex dynamic systems model of dyadic play was used to make predictions with regard to duration and number of contact-episodes during play of same-sex dyads, both on the micro- (i.e., per individual session), meso- (i.e., in smoothed data), and macro time scale (i.e., the change over six consecutive play sessions). The empirical data came from a study that examined the collaborative play skills of children who experienced six twenty minute play sessions within a three week period of time. Monte Carlo permutation analyses were used to compare model predictions and empirical data. The findings point to strongly asymmetric distributions in the duration and number of contact episodes in all dyads over the six sessions, as a direct consequence of the underlying dynamics of the play system. The model prediction that girls-dyads would show longer contact episodes than boys-dyads was confirmed, but the prediction regarding the difference in number of peaks was not confirmed. In addition, the majority of the model predictions regarding changes over the course of six sessions were consistent with the data. That is, the average duration and the maximum duration of contact-episodes increases both in boys-dyads and girls-dyads, but differences occur in the strength of the increase. Contrary to expectation, the number of contact-episodes decreases both in boys-dyads and in girls-dyads. PMID:24894265

  1. Copper complexation screen reveals compounds with potent antibiotic properties against methicillin-resistant Staphylococcus aureus.

    PubMed

    Haeili, Mehri; Moore, Casey; Davis, Christopher J C; Cochran, James B; Shah, Santosh; Shrestha, Tej B; Zhang, Yaofang; Bossmann, Stefan H; Benjamin, William H; Kutsch, Olaf; Wolschendorf, Frank

    2014-07-01

    Macrophages take advantage of the antibacterial properties of copper ions in the killing of bacterial intruders. However, despite the importance of copper for innate immune functions, coordinated efforts to exploit copper ions for therapeutic interventions against bacterial infections are not yet in place. Here we report a novel high-throughput screening platform specifically developed for the discovery and characterization of compounds with copper-dependent antibacterial properties toward methicillin-resistant Staphylococcus aureus (MRSA). We detail how one of the identified compounds, glyoxal-bis(N4-methylthiosemicarbazone) (GTSM), exerts its potent strictly copper-dependent antibacterial properties on MRSA. Our data indicate that the activity of the GTSM-copper complex goes beyond the general antibacterial effects of accumulated copper ions and suggest that, in contrast to prevailing opinion, copper complexes can indeed exhibit species- and target-specific activities. Based on experimental evidence, we propose that copper ions impose structural changes upon binding to the otherwise inactive GTSM ligand and transfer antibacterial properties to the chelate. In turn, GTSM determines target specificity and utilizes a redox-sensitive release mechanism through which copper ions are deployed at or in close proximity to a putative target. According to our proof-of-concept screen, copper activation is not a rare event and even extends to already established drugs. Thus, copper-activated compounds could define a novel class of anti-MRSA agents that amplify copper-dependent innate immune functions of the host. To this end, we provide a blueprint for a high-throughput drug screening campaign which considers the antibacterial properties of copper ions at the host-pathogen interface. PMID:24752262

  2. Structural Identification of the Vps18 β-Propeller Reveals a Critical Role in the HOPS Complex Stability and Function*

    PubMed Central

    Behrmann, Heide; Lürick, Anna; Kuhlee, Anne; Balderhaar, Henning Kleine; Bröcker, Cornelia; Kümmel, Daniel; Engelbrecht-Vandré, Siegfried; Gohlke, Ulrich; Raunser, Stefan; Heinemann, Udo; Ungermann, Christian

    2014-01-01

    Membrane fusion at the vacuole, the lysosome equivalent in yeast, requires the HOPS tethering complex, which is recruited by the Rab7 GTPase Ypt7. HOPS provides a template for the assembly of SNAREs and thus likely confers fusion at a distinct position on vacuoles. Five of the six subunits in HOPS have a similar domain prediction with strong similarity to COPII subunits and nuclear porins. Here, we show that Vps18 indeed has a seven-bladed β-propeller as its N-terminal domain by revealing its structure at 2.14 Å. The Vps18 N-terminal domain can interact with the N-terminal part of Vps11 and also binds to lipids. Although deletion of the Vps18 N-terminal domain does not preclude HOPS assembly, as revealed by negative stain electron microscopy, the complex is instable and cannot support membrane fusion in vitro. We thus conclude that the β-propeller of Vps18 is required for HOPS stability and function and that it can serve as a starting point for further structural analyses of the HOPS tethering complex. PMID:25324549

  3. BstYI bound to noncognate DNA reveals a "hemispecific" complex: implications for DNA scanning.

    PubMed

    Townson, Sharon A; Samuelson, James C; Bao, Yongming; Xu, Shuang-Yong; Aggarwal, Aneel K

    2007-04-01

    DNA recognition by proteins is essential for specific expression of genes in a living organism. En route to a target DNA site, a protein will often sample noncognate DNA sites through nonspecific protein-DNA interactions, resulting in a variety of conformationally different binding states. We present here the crystal structure of endonuclease BstYI bound to a noncognate DNA. Surprisingly, the structure reveals the enzyme in a "hemispecific" binding state on the pathway between nonspecific and specific recognition. A single base pair change in the DNA abolishes binding of only one monomer, with the second monomer bound specifically. We show that the enzyme binds essentially as a rigid body, and that one end of the DNA is accommodated loosely in the binding cleft while the other end is held tightly. Another intriguing feature of the structure is Ser172, which has a dual role in establishing nonspecific and specific contacts. Taken together, the structure provides a snapshot of an enzyme in a "paused" intermediate state that may be part of a more general mechanism of scanning DNA. PMID:17437717

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

    PubMed Central

    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 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. PMID:25565106

  5. Genome sequencing reveals complex secondary metabolome in the marine actinomycete Salinispora tropica

    PubMed Central

    Udwary, Daniel W.; Zeigler, Lisa; Asolkar, Ratnakar N.; Singan, Vasanth; Lapidus, Alla; Fenical, William; Jensen, Paul R.; Moore, Bradley S.

    2007-01-01

    Recent fermentation studies have identified actinomycetes of the marine-dwelling genus Salinispora as prolific natural product producers. To further evaluate their biosynthetic potential, we sequenced the 5,183,331-bp S. tropica CNB-440 circular genome and analyzed all identifiable secondary natural product gene clusters. Our analysis shows that S. tropica dedicates a large percentage of its genome (≈9.9%) to natural product assembly, which is greater than previous Streptomyces genome sequences as well as other natural product-producing actinomycetes. The S. tropica genome features polyketide synthase systems of every known formally classified family, nonribosomal peptide synthetases, and several hybrid clusters. Although a few clusters appear to encode molecules previously identified in Streptomyces species, the majority of the 17 biosynthetic loci are novel. Specific chemical information about putative and observed natural product molecules is presented and discussed. In addition, our bioinformatic analysis not only was critical for the structure elucidation of the polyene macrolactam salinilactam A, but its structural analysis aided the genome assembly of the highly repetitive slm loci. This study firmly establishes the genus Salinispora as a rich source of drug-like molecules and importantly reveals the powerful interplay between genomic analysis and traditional natural product isolation studies. PMID:17563368

  6. First DNA sequences from Asian cave bear fossils reveal deep divergences and complex phylogeographic patterns.

    PubMed

    Knapp, Michael; Rohland, Nadin; Weinstock, Jacobo; Baryshnikov, Gennady; Sher, Andrei; Nagel, Doris; Rabeder, Gernot; Pinhasi, Ron; Schmidt, Heiko A; Hofreiter, Michael

    2009-03-01

    Until recently, cave bears were believed to have only inhabited Europe. However, recent morphological evidence suggests that cave bears' geographic range extended as far east as Transbaikalia, Eastern Siberia. These Asian cave bears were morphologically distinct from European cave bears. However, how they related to European lineages remains unclear, stressing the need to assess the phylogenetic and phylogeographic relationship between Asian cave bears and their European relatives. In this work, we address this issue using a 227 base-pair fragment of the mitochondrial control region obtained from nine fossil bone samples from eight sites from the Urals, Caucasus, Altai Mountains, Ukraine and Yana River region in Eastern Siberia. Results of the phylogenetic analyses indicate that (i) the cave bear from the Yana River is most closely related to cave bears from the Caucasus region; (ii) the Caucasus/Yana group of bears is genetically very distinct from both European cave bears and brown bears, suggesting that these bears could represent an independent species; and (iii) the Western European cave bear lineage reached at least temporarily to the Altai Mountains, 7000 km east of their known centre of distribution. These results suggest that the diversity of cave bears was greater than previously believed, and that they could survive in a much wider range of ecological conditions than previously assumed. They also agree with recent studies on other extinct and extant species, such as wolves, hyenas and steppe bison, which have also revealed higher genetic and ecological diversity in Pleistocene populations than previously known. PMID:19226321

  7. Ethiopian Genetic Diversity Reveals Linguistic Stratification and Complex Influences on the Ethiopian Gene Pool

    PubMed Central

    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

    2012-01-01

    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. PMID:22726845

  8. Global mapping of herpesvirus-host protein complexes reveals a novel transcription strategy for late genes

    PubMed Central

    Davis, Zoe H.; Verschueren, Erik; Jang, Gwendolyn M.; Kleffman, Kevin; Johnson, Jeffrey R.; Park, Jimin; Von Dollen, John; Maher, M. Cyrus; Johnson, Tasha; Newton, William; Jäger, Stefanie; Shales, Michael; Horner, Julie; Hernandez, Ryan D.; Krogan, Nevan J.; Glaunsinger, Britt A.

    2014-01-01

    SUMMARY Mapping host-pathogen interactions has proven instrumental for understanding how viruses manipulate host machinery and how numerous cellular processes are regulated. DNA viruses such as herpesviruses have relatively large coding capacity and thus can target an extensive network of cellular proteins. To identify the host proteins hijacked by this pathogen, we systematically affinity tagged and purified all 89 proteins of Kaposi’s sarcoma-associated herpesvirus (KSHV) from human cells. Mass spectrometry of this material identified over 500 virus-host interactions. KSHV causes AIDS-associated cancers and its interaction network is enriched for proteins linked to cancer and overlaps with proteins that are also targeted by HIV-1. We found that the conserved KSHV protein ORF24 binds to RNA polymerase II and brings it to viral late promoters by mimicking and replacing cellular TATA-box-binding protein (TBP). This is required for herpesviral late gene expression, a complex and poorly understood phase of the viral lifecycle. PMID:25544563

  9. Crystal structure of equine serum albumin in complex with cetirizine reveals a novel drug binding site.

    PubMed

    Handing, Katarzyna B; Shabalin, Ivan G; Szlachta, Karol; Majorek, Karolina A; Minor, Wladek

    2016-03-01

    Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1Å. Cetirizine is bound in two sites--a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizine binding sites. We show that the residues forming the binding pockets in ESA are highly conserved in human serum albumin (HSA), and suggest that binding of cetirizine to HSA will be similar. In support of that hypothesis, we show that the dissociation constants for cetirizine binding to CBS2 in ESA and HSA are identical using tryptophan fluorescence quenching. Presence of lysine and arginine residues that have been previously reported to undergo nonenzymatic glycosylation in CBS1 and CBS2 suggests that cetirizine transport in patients with diabetes could be altered. A review of all available SA structures from the PDB shows that in addition to the novel drug binding site we present here (CBS1), there are two pockets on SA capable of binding drugs that do not overlap with fatty acid binding sites and have not been discussed in published reviews. PMID:26896718

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

    PubMed

    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. PMID:26179888

  11. Transcriptome analysis of mammary tissues reveals complex patterns of transporter gene expression during pregnancy and lactation.

    PubMed

    Anantamongkol, Utchariya; Charoenphandhu, Narattaphol; Wongdee, Kannikar; Teerapornpuntakit, Jarinthorn; Suthiphongchai, Tuangporn; Prapong, Siriwan; Krishnamra, Nateetip

    2010-01-01

    As a complex Ca2+-rich fluid mixture of water, casein, lactose and several ions, milk secretion requires a number of unknown transporters, which can be identified by a genome-wide microarray study in mammary tissues of lactating animals. Ca2+ was reported to be secreted across mammary epithelial cells through the transcellular pathway, presumably involving TRPC (canonical transient receptor potential) channels. In the present study, we have used quantitative real-time PCR to demonstrate that the human mammary cell line MCF-7, as well as rat mammary tissues from pregnant and lactating rats, expressed TRPC1, TRPC5 and TRPC6. Expression of TRPC1, TRPC5 and TRPC7 were markedly up-regulated, whereas that of TRPC3 and TRPC4 was down-regulated in the early lactating period. To further identify other transporter genes affected by lactation, a highly sensitive Illumina microarray featuring Bead Array technology was performed on RNA samples from mammary tissues of lactating rats. We found that, of the 384 transcripts changed during lactation, 31 transcripts were involved in the transport of water and electrolytes, such as Ca2+, Na+, K+, Cl-, I-, Fe2+, sulfate and phosphate. The present study, therefore, provides information for further investigation of the mechanism of lactation-induced transport adaptation in mammary epithelial cells. PMID:19947944

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

    SciTech Connect

    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.

  13. New Levels of Language Processing Complexity and Organization Revealed by Granger Causation

    PubMed Central

    Gow, David W.; Caplan, David N.

    2012-01-01

    Granger causation analysis of high spatiotemporal resolution reconstructions of brain activation offers a new window on the dynamic interactions between brain areas that support language processing. Premised on the observation that causes both precede and uniquely predict their effects, this approach provides an intuitive, model-free means of identifying directed causal interactions in the brain. It requires the analysis of all non-redundant potentially interacting signals, and has shown that even “early” processes such as speech perception involve interactions of many areas in a strikingly large network that extends well beyond traditional left hemisphere perisylvian cortex that play out over hundreds of milliseconds. In this paper we describe this technique and review several general findings that reframe the way we think about language processing and brain function in general. These include the extent and complexity of language processing networks, the central role of interactive processing dynamics, the role of processing hubs where the input from many distinct brain regions are integrated, and the degree to which task requirements and stimulus properties influence processing dynamics and inform our understanding of “language-specific” localized processes. PMID:23293611

  14. Physical Conditions of Eta Car Complex Environment Revealed From Photoionization Modeling

    NASA Technical Reports Server (NTRS)

    Verner, E. M.; Bruhweiler, F.; Nielsen, K. E.; Gull, T.; Kober, G. Vieira; Corcoran, M.

    2006-01-01

    The very massive star, Eta Carinae, is enshrouded in an unusual complex environment of nebulosities and structures. The circumstellar gas gives rise to distinct absorption and emission components at different velocities and distances from the central source(s). Through photoionization modeling, we find that the radiation field from the more massive B-star companion supports the low ionization structure throughout the 5.54 year period. The radiation field of an evolved O-star is required to produce the higher ionization . emission seen across the broad maximum. Our studies utilize the HST/STIS data and model calculations of various regimes from doubly ionized species (T= 10,000K) to the low temperature (T = 760 K) conditions conductive to molecule formation (CH and OH). Overall analysis suggests the high depletion in C and O and the enrichment in He and N. The sharp molecular and ionic absorptions in this extensively CNO - processed material offers a unique environment for studying the chemistry, dust formation processes, and nucleosynthesis in the ejected layers of a highly evolved massive star.

  15. Complex patterns of mitochondrial dynamics in human pancreatic cells revealed by fluorescent confocal imaging.

    PubMed

    Kuznetsov, Andrey V; Hermann, Martin; Troppmair, Jakob; Margreiter, Raimund; Hengster, Paul

    2010-01-01

    Mitochondrial morphology and intracellular organization are tightly controlled by the processes of mitochondrial fission-fusion. Moreover, mitochondrial movement and redistribution provide a local ATP supply at cellular sites of particular demands. Here we analysed mitochondrial dynamics in isolated primary human pancreatic cells. Using real time confocal microscopy and mitochondria-specific fluorescent probes tetramethylrhodamine methyl ester and MitoTracker Green we documented complex and novel patterns of spatial and temporal organization of mitochondria, mitochondrial morphology and motility. The most commonly observed types of mitochondrial dynamics were (i) fast fission and fusion; (ii) small oscillating movements of the mitochondrial network; (iii) larger movements, including filament extension, retraction, fast (0.1-0.3 mum/sec.) and frequent oscillating (back and forth) branching in the mitochondrial network; (iv) as well as combinations of these actions and (v) long-distance intracellular translocation of single spherical mitochondria or separated mitochondrial filaments with velocity up to 0.5 mum/sec. Moreover, we show here for the first time, a formation of unusual mitochondrial shapes like rings, loops, and astonishingly even knots created from one or more mitochondrial filaments. These data demonstrate the presence of extensive heterogeneity in mitochondrial morphology and dynamics in living cells under primary culture conditions. In summary, this study reports new patterns of morphological changes and dynamic motion of mitochondria in human pancreatic cells, suggesting an important role of integrations of mitochondria with other intracellular structures and systems. PMID:19382913

  16. Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins

    PubMed Central

    Lavy, Meirav; Prigge, Michael J; Tao, Sibo; Shain, Stephanie; Kuo, April; Kirchsteiger, Kerstin; Estelle, Mark

    2016-01-01

    The coordinated action of the auxin-sensitive Aux/IAA transcriptional repressors and ARF transcription factors produces complex gene-regulatory networks in plants. Despite their importance, our knowledge of these two protein families is largely based on analysis of stabilized forms of the Aux/IAAs, and studies of a subgroup of ARFs that function as transcriptional activators. To understand how auxin regulates gene expression we generated a Physcomitrella patens line that completely lacks Aux/IAAs. Loss of the repressors causes massive changes in transcription with misregulation of over a third of the annotated genes. Further, we find that the aux/iaa mutant is blind to auxin indicating that auxin regulation of transcription occurs exclusively through Aux/IAA function. We used the aux/iaa mutant as a simplified platform for studies of ARF function and demonstrate that repressing ARFs regulate auxin-induced genes and fine-tune their expression. Further the repressing ARFs coordinate gene induction jointly with activating ARFs and the Aux/IAAs. DOI: http://dx.doi.org/10.7554/eLife.13325.001 PMID:27247276

  17. Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins.

    PubMed

    Lavy, Meirav; Prigge, Michael J; Tao, Sibo; Shain, Stephanie; Kuo, April; Kirchsteiger, Kerstin; Estelle, Mark

    2016-01-01

    The coordinated action of the auxin-sensitive Aux/IAA transcriptional repressors and ARF transcription factors produces complex gene-regulatory networks in plants. Despite their importance, our knowledge of these two protein families is largely based on analysis of stabilized forms of the Aux/IAAs, and studies of a subgroup of ARFs that function as transcriptional activators. To understand how auxin regulates gene expression we generated a Physcomitrella patens line that completely lacks Aux/IAAs. Loss of the repressors causes massive changes in transcription with misregulation of over a third of the annotated genes. Further, we find that the aux/iaa mutant is blind to auxin indicating that auxin regulation of transcription occurs exclusively through Aux/IAA function. We used the aux/iaa mutant as a simplified platform for studies of ARF function and demonstrate that repressing ARFs regulate auxin-induced genes and fine-tune their expression. Further the repressing ARFs coordinate gene induction jointly with activating ARFs and the Aux/IAAs. PMID:27247276

  18. Environmental Interactions and Epistasis Are Revealed in the Proteomic Responses to Complex Stimuli

    PubMed Central

    Samir, Parimal; Rahul; Slaughter, James C.; Link, Andrew J.

    2015-01-01

    Ultimately, the genotype of a cell and its interaction with the environment determine the cell’s biochemical state. While the cell’s response to a single stimulus has been studied extensively, a conceptual framework to model the effect of multiple environmental stimuli applied concurrently is not as well developed. In this study, we developed the concepts of environmental interactions and epistasis to explain the responses of the S. cerevisiae proteome to simultaneous environmental stimuli. We hypothesize that, as an abstraction, environmental stimuli can be treated as analogous to genetic elements. This would allow modeling of the effects of multiple stimuli using the concepts and tools developed for studying gene interactions. Mirroring gene interactions, our results show that environmental interactions play a critical role in determining the state of the proteome. We show that individual and complex environmental stimuli behave similarly to genetic elements in regulating the cellular responses to stimuli, including the phenomena of dominance and suppression. Interestingly, we observed that the effect of a stimulus on a protein is dominant over other stimuli if the response to the stimulus involves the protein. Using publicly available transcriptomic data, we find that environmental interactions and epistasis regulate transcriptomic responses as well. PMID:26247773

  19. Dynamic Modelling Reveals ‘Hotspots’ on the Pathway to Enzyme-Substrate Complex Formation

    PubMed Central

    Gordon, Shane E.; Weber, Daniel K.; Downton, Matthew T.; Wagner, John; Perugini, Matthew A.

    2016-01-01

    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. PMID:26967332

  20. Enrichment of SNPs in Functional Categories Reveals Genes Affecting Complex Traits.

    PubMed

    Zhao, Huiying; Fan, Dongsheng; Nyholt, Dale R; Yang, Yuedong

    2016-08-01

    Genome-wide association studies (GWAS) have indicated potential to identify heritability of common complex phenotypes, but traditional approaches have limited ability to detect hiding signals because single SNP has weak effect size accounting for only a small fraction of overall phenotypic variations. To improve the power of GWAS, methods have been developed to identify truly associated genes by jointly testing effects of all SNPs. However, equally considering all SNPs within a gene might dilute strong signals of SNPs in real functional categories. Here, we observed a consistent pattern on enrichment of significant SNPs in eight functional categories across six phenotypes, with the highest enrichment in coding and both UTR regions while the lowest enrichment in the intron. Based on the pattern of SNP enrichment in functional categories, we developed a new approach for detecting gene associations on traits (DGAT) by selecting the most significant functional category and then using SNPs within it to assess gene associations. The method was found to be robust in type I error rate on simulated data, and to have mostly higher power in detecting associated genes for three different diseases than other methods. Further analysis indicated ability of the DGAT to detect novel genes. The DGAT is available by http://sparks-lab.org/server/DGAT. PMID:27113629

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

    PubMed

    Langereis, Martijn A; Bakkers, Mark J G; Deng, Lingquan; Padler-Karavani, Vered; Vervoort, Stephin J; Hulswit, Ruben J G; van Vliet, Arno L W; Gerwig, Gerrit J; de Poot, Stefanie A H; Boot, Willemijn; van Ederen, Anne Marie; Heesters, Balthasar A; van der Loos, Chris M; van Kuppeveld, Frank J M; Yu, Hai; Huizinga, Eric G; Chen, Xi; Varki, Ajit; Kamerling, Johannis P; de Groot, Raoul J

    2015-06-30

    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. PMID:26095364

  2. Proteome-wide analysis reveals widespread lysine acetylation of major protein complexes in the malaria parasite

    PubMed Central

    Cobbold, Simon A.; Santos, Joana M.; Ochoa, Alejandro; Perlman, David H.; Llinás, Manuel

    2016-01-01

    Lysine acetylation is a ubiquitous post-translational modification in many organisms including the malaria parasite Plasmodium falciparum, yet the full extent of acetylation across the parasite proteome remains unresolved. Moreover, the functional significance of acetylation or how specific acetyl-lysine sites are regulated is largely unknown. Here we report a seven-fold expansion of the known parasite ‘acetylome’, characterizing 2,876 acetylation sites on 1,146 proteins. We observe that lysine acetylation targets a diverse range of protein complexes and is particularly enriched within the Apicomplexan AP2 (ApiAP2) DNA-binding protein family. Using quantitative proteomics we determined that artificial perturbation of the acetate/acetyl-CoA balance alters the acetyl-lysine occupancy of several ApiAP2 DNA-binding proteins and related transcriptional proteins. This metabolic signaling could mediate significant downstream transcriptional responses, as we show that acetylation of an ApiAP2 DNA-binding domain ablates its DNA-binding propensity. Lastly, we investigated the acetyl-lysine targets of each class of lysine deacetylase in order to begin to explore how each class of enzyme contributes to regulating the P. falciparum acetylome. PMID:26813983

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

    PubMed

    Gordon, Shane E; Weber, Daniel K; Downton, Matthew T; Wagner, John; Perugini, Matthew A

    2016-03-01

    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. PMID:26967332

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

    PubMed

    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. PMID:26561038

  5. A Quantitative Characterization of Nucleoplasmin/Histone Complexes Reveals Chaperone Versatility.

    PubMed

    Fernández-Rivero, Noelia; Franco, Aitor; Velázquez-Campoy, Adrian; Alonso, Edurne; Muga, Arturo; Prado, Adelina

    2016-01-01

    Nucleoplasmin (NP) is an abundant histone chaperone in vertebrate oocytes and embryos involved in storing and releasing maternal histones to establish and maintain the zygotic epigenome. NP has been considered a H2A-H2B histone chaperone, and recently it has been shown that it can also interact with H3-H4. However, its interaction with different types of histones has not been quantitatively studied so far. We show here that NP binds H2A-H2B, H3-H4 and linker histones with Kd values in the subnanomolar range, forming different complexes. Post-translational modifications of NP regulate exposure of the polyGlu tract at the disordered distal face of the protein and induce an increase in chaperone affinity for all histones. The relative affinity of NP for H2A-H2B and linker histones and the fact that they interact with the distal face of the chaperone could explain their competition for chaperone binding, a relevant process in NP-mediated sperm chromatin remodelling during fertilization. Our data show that NP binds H3-H4 tetramers in a nucleosomal conformation and dimers, transferring them to DNA to form disomes and tetrasomes. This finding might be relevant to elucidate the role of NP in chromatin disassembly and assembly during replication and transcription. PMID:27558753

  6. Transcriptome and Secretome Analyses of Phanerochaete chrysosporium Reveal Complex Patterns of Gene Expression▿ †

    PubMed Central

    Vanden Wymelenberg, Amber; Gaskell, Jill; Mozuch, Mike; Kersten, Phil; Sabat, Grzegorz; Martinez, Diego; Cullen, Dan

    2009-01-01

    The wood decay basidiomycete Phanerochaete chrysosporium was grown under standard ligninolytic or cellulolytic conditions and subjected to whole-genome expression microarray analysis and liquid chromatography-tandem mass spectrometry of extracellular proteins. A total of 545 genes were flagged on the basis of significant changes in transcript accumulation and/or peptide sequences of the secreted proteins. Under nitrogen or carbon limitation, lignin and manganese peroxidase expression increased relative to nutrient replete medium. Various extracellular oxidases were also secreted in these media, supporting a physiological connection based on peroxide generation. Numerous genes presumed to be involved in mobilizing and recycling nitrogen were expressed under nitrogen limitation, and among these were several secreted glutamic acid proteases not previously observed. In medium containing microcrystalline cellulose as the sole carbon source, numerous genes encoding carbohydrate-active enzymes were upregulated. Among these were six members of the glycoside hydrolase family 61, as well as several polysaccharide lyases and carbohydrate esterases. Presenting a daunting challenge for future research, more than 190 upregulated genes are predicted to encode proteins of unknown function. Of these hypothetical proteins, approximately one-third featured predicted secretion signals, and 54 encoded proteins detected in extracellular filtrates. Our results affirm the importance of certain oxidative enzymes and, underscoring the complexity of lignocellulose degradation, also support an important role for many new proteins of unknown function. PMID:19376920

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

    PubMed Central

    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. PMID:26561038

  8. A Quantitative Characterization of Nucleoplasmin/Histone Complexes Reveals Chaperone Versatility

    PubMed Central

    Fernández-Rivero, Noelia; Franco, Aitor; Velázquez-Campoy, Adrian; Alonso, Edurne; Muga, Arturo; Prado, Adelina

    2016-01-01

    Nucleoplasmin (NP) is an abundant histone chaperone in vertebrate oocytes and embryos involved in storing and releasing maternal histones to establish and maintain the zygotic epigenome. NP has been considered a H2A–H2B histone chaperone, and recently it has been shown that it can also interact with H3-H4. However, its interaction with different types of histones has not been quantitatively studied so far. We show here that NP binds H2A–H2B, H3-H4 and linker histones with Kd values in the subnanomolar range, forming different complexes. Post-translational modifications of NP regulate exposure of the polyGlu tract at the disordered distal face of the protein and induce an increase in chaperone affinity for all histones. The relative affinity of NP for H2A–H2B and linker histones and the fact that they interact with the distal face of the chaperone could explain their competition for chaperone binding, a relevant process in NP-mediated sperm chromatin remodelling during fertilization. Our data show that NP binds H3-H4 tetramers in a nucleosomal conformation and dimers, transferring them to DNA to form disomes and tetrasomes. This finding might be relevant to elucidate the role of NP in chromatin disassembly and assembly during replication and transcription. PMID:27558753

  9. SerRS-tRNASec complex structures reveal mechanism of the first step in selenocysteine biosynthesis.

    PubMed

    Wang, Caiyan; Guo, Yu; Tian, Qingnan; Jia, Qian; Gao, Yuanzhu; Zhang, Qinfen; Zhou, Chun; Xie, Wei

    2015-12-01

    Selenocysteine (Sec) is found in the catalytic centers of many selenoproteins and plays important roles in living organisms. Malfunctions of selenoproteins lead to various human disorders including cancer. Known as the 21st amino acid, the biosynthesis of Sec involves unusual pathways consisting of several stages. While the later stages of the pathways are well elucidated, the molecular basis of the first stage-the serylation of Sec-specific tRNA (tRNA(Sec)) catalyzed by seryl-tRNA synthetase (SerRS)-is unclear. Here we present two cocrystal structures of human SerRS bound with tRNA(Sec) in different stoichiometry and confirm the formation of both complexes in solution by various characterization techniques. We discovered that the enzyme mainly recognizes the backbone of the long variable arm of tRNA(Sec) with few base-specific contacts. The N-terminal coiled-coil region works like a long-range lever to precisely direct tRNA 3' end to the other protein subunit for aminoacylation in a conformation-dependent manner. Restraints of the flexibility of the coiled-coil greatly reduce serylation efficiencies. Lastly, modeling studies suggest that the local differences present in the D- and T-regions as well as the characteristic U20:G19:C56 base triple in tRNA(Sec) may allow SerRS to distinguish tRNA(Sec) from closely related tRNA(Ser) substrate. PMID:26433229

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

    PubMed

    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 (<150 kg) ungulates and were relatively moderate (change of 4 individuals across all habitats). In comparison, ungulate aggregations tended to increase at a slightly lower rate in habitat that was more open. The 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26134169

  12. Complex Regulation Pattern of IRF3 Activation Revealed by a Novel Dimerization Reporter System.

    PubMed

    Wang, Zining; Ji, Jingyun; Peng, Di; Ma, Feng; Cheng, Genhong; Qin, F Xiao-Feng

    2016-05-15

    Induction of type I IFN (IFN-I) is essential for host antiviral immune responses. However, IFN-I also plays divergent roles in antibacterial immunity, persistent viral infections, autoimmune diseases, and tumorigenesis. IFN regulatory factor 3 (IRF3) is the master transcription factor that controls IFN-I production via phosphorylation-dependent dimerization in most cell types in response to viral infections and various innate stimuli by pathogen-associated molecular patterns (PAMPs). To monitor the dynamic process of IRF3 activation, we developed a novel IRF3 dimerization reporter based on bimolecular luminescence complementation (BiLC) techniques, termed the IRF3-BiLC reporter. Robust induction of luciferase activity of the IRF3-BiLC reporter was observed upon viral infection and PAMP stimulation with a broad dynamic range. Knockout of TANK-binding kinase 1, the critical upstream kinase of IRF3, as well as the mutation of serine 386, the essential phosphorylation site of IRF3, completely abolished the luciferase activity of IRF3-BiLC reporter, confirming the authenticity of IRF3 activation. Taken together, these results demonstrated that the IRF3-BiLC reporter is a highly specific, reliable, and sensitive system to measure IRF3 activity. Using this reporter system, we further observed that the temporal pattern and magnitude of IRF3 activation induced by various PAMPs are highly complex with distinct cell type-specific characteristics, and IRF3 dimerization is a direct regulatory node for IFN-α/β receptor-mediated feed-forward regulation and crosstalk with other pathways. Therefore, the IRF3-BiLC reporter has multiple potential applications, including mechanistic studies as well as the identification of novel compounds that can modulate IRF3 activation. PMID:27045107

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

    PubMed

    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. PMID:27569545

  14. Genomic analysis of the immune gene repertoire of amphioxus reveals extraordinary innate complexity and diversity

    PubMed Central

    Huang, Shengfeng; Yuan, Shaochun; Guo, Lei; Yu, Yanhong; Li, Jun; Wu, Tao; Liu, Tong; Yang, Manyi; Wu, Kui; Liu, Huiling; Ge, Jin; Yu, Yingcai; Huang, Huiqing; Dong, Meiling; Yu, Cuiling; Chen, Shangwu; Xu, Anlong

    2008-01-01

    It has been speculated that before vertebrates evolved somatic diversity-based adaptive immunity, the germline-encoded diversity of innate immunity may have been more developed. Amphioxus occupies the basal position of the chordate phylum and hence is an important reference to the evolution of vertebrate immunity. Here we report the first comprehensive genomic survey of the immune gene repertoire of the amphioxus Branchiostoma floridae. It has been reported that the purple sea urchin has a vastly expanded innate receptor repertoire not previously seen in other species, which includes 222 toll-like receptors (TLRs), 203 NOD/NALP-like receptors (NLRs), and 218 scavenger receptors (SRs). We discovered that the amphioxus genome contains comparable expansion with 71 TLR gene models, 118 NLR models, and 270 SR models. Amphioxus also expands other receptor-like families, including 1215 C-type lectin models, 240 LRR and IGcam-containing models, 1363 other LRR-containing models, 75 C1q-like models, 98 ficolin-like models, and hundreds of models containing complement-related domains. The expansion is not restricted to receptors but is likely to extend to intermediate signal transducers because there are 58 TIR adapter-like models, 36 TRAF models, 44 initiator caspase models, and 541 death-fold domain-containing models in the genome. Amphioxus also has a sophisticated TNF system and a complicated complement system not previously seen in other invertebrates. Besides the increase of gene number, domain combinations of immune proteins are also increased. Altogether, this survey suggests that the amphioxus, a species without vertebrate-type adaptive immunity, holds extraordinary innate complexity and diversity. PMID:18562681

  15. Genomic analysis of the immune gene repertoire of amphioxus reveals extraordinary innate complexity and diversity.

    PubMed

    Huang, Shengfeng; Yuan, Shaochun; Guo, Lei; Yu, Yanhong; Li, Jun; Wu, Tao; Liu, Tong; Yang, Manyi; Wu, Kui; Liu, Huiling; Ge, Jin; Yu, Yingcai; Huang, Huiqing; Dong, Meiling; Yu, Cuiling; Chen, Shangwu; Xu, Anlong

    2008-07-01

    It has been speculated that before vertebrates evolved somatic diversity-based adaptive immunity, the germline-encoded diversity of innate immunity may have been more developed. Amphioxus occupies the basal position of the chordate phylum and hence is an important reference to the evolution of vertebrate immunity. Here we report the first comprehensive genomic survey of the immune gene repertoire of the amphioxus Branchiostoma floridae. It has been reported that the purple sea urchin has a vastly expanded innate receptor repertoire not previously seen in other species, which includes 222 toll-like receptors (TLRs), 203 NOD/NALP-like receptors (NLRs), and 218 scavenger receptors (SRs). We discovered that the amphioxus genome contains comparable expansion with 71 TLR gene models, 118 NLR models, and 270 SR models. Amphioxus also expands other receptor-like families, including 1215 C-type lectin models, 240 LRR and IGcam-containing models, 1363 other LRR-containing models, 75 C1q-like models, 98 ficolin-like models, and hundreds of models containing complement-related domains. The expansion is not restricted to receptors but is likely to extend to intermediate signal transducers because there are 58 TIR adapter-like models, 36 TRAF models, 44 initiator caspase models, and 541 death-fold domain-containing models in the genome. Amphioxus also has a sophisticated TNF system and a complicated complement system not previously seen in other invertebrates. Besides the increase of gene number, domain combinations of immune proteins are also increased. Altogether, this survey suggests that the amphioxus, a species without vertebrate-type adaptive immunity, holds extraordinary innate complexity and diversity. PMID:18562681

  16. Species delimitation without prior knowledge: DISSECT reveals extensive cryptic speciation in the Silene aegyptiaca complex (Caryophyllaceae).

    PubMed

    Toprak, Zeynep; Pfeil, Bernard E; Jones, Graham; Marcussen, Thomas; Ertekin, Alaattin Selçuk; Oxelman, Bengt

    2016-09-01

    Species delimitation is a major focus of biosystematics. In recent years, considerable progress has been achieved with the development of the multispecies coalescent (MSC) model, where species constitute the branches of the species tree or network. However, researchers are faced with the limitation that the MSC method of choice often requires a priori assignment of individuals to species. This not only introduces subjectivitiy into the analyses, but may also lead to meaningless species tree hypotheses, if the allele-to-species assignments are inaccurate. DISSECT is a recently introduced method that does not require a priori allele-to-species assignments, but instead examines the posterior probabilities of groupings (clusterings) of individuals under study. Using the DISSECT approach, we analysed genetic data from 75 individual plants belonging to the Silene aegyptiaca species complex that has previously been divided into 3-5 species. Marginal likelihood estimates from (*)BEAST analyses, run with predefined species classifications, strongly favour those compatible with the DISSECT result over those from morphology- and geography-based taxonomy. We found at least nine species, including several cryptic ones, for which no clear geographical or morphological patterns are correlated. However, the limited data and the possibility of unmodelled processes mean there is still much uncertainty about the true number of MSC species, and for taxonomic purposes, other criteria might be relevant. Nevertheless, we argue that the approach signifies an important step towards objective and testable species delimitations in any organismal group. In particular, it makes it possible to avoid biologically irrelevant species classifications. PMID:27233442

  17. PKA RIα Homodimer Structure Reveals an Intermolecular Interface with Implications for Cooperative cAMP Binding and Carney Complex Disease

    PubMed Central

    Bruystens, Jessica G.H.; Wu, Jian; Fortezzo, Audrey; Kornev, Alexandr P.; Blumenthal, Donald K.; Taylor, Susan S.

    2014-01-01

    Summary The regulatory (R) subunit is the cAMP receptor of protein kinase A. Following cAMP binding, the inactive PKA holoenzyme complex separates into two active catalytic (C) subunits and a cAMP-bound R dimer. Thus far, only monomeric R structures have been solved, which fell short in explaining differences of cAMP binding for the full-length protein as compared to the truncated R subunits. Here we solved a full-length R-dimer structure that reflects the biologically relevant conformation, and this structure agrees well with small angle X-ray scattering. An isoform-specific interface is revealed between the protomers. This interface acts as an intermolecular sensor for cAMP and explains the cooperative character of cAMP binding to the RIα dimer. Mutagenesis of residues on this interface not only leads to structural and biochemical changes, but is also linked to Carney complex disease. PMID:24316401

  18. Structure-function analyses of the human SIX1-EYA2 complex reveal insights into metastasis and BOR syndrome

    SciTech Connect

    Patrick, Aaron N.; Cabrera, Joshua H.; Smith, Anna L.; Chen, Xiaojiang S.; Ford, Heide L.; Zhao, Rui

    2013-05-06

    SIX1 interacts with EYA to form a bipartite transcription factor essential for mammalian development. Loss of function of this complex causes branchio-oto-renal (BOR) syndrome, whereas re-expression of SIX1 or EYA promotes metastasis. Here we describe the 2.0-Å structure of SIX1 bound to EYA2, which suggests a new DNA-binding mechanism for SIX1 and provides a rationale for the effect of BOR syndrome mutations. The structure also reveals that SIX1 uses predominantly a single helix to interact with EYA. Substitution of a single amino acid in this helix is sufficient to disrupt SIX1-EYA interaction, SIX1-mediated epithelial-mesenchymal transition and metastasis in mouse models. Given that SIX1 and EYA are overexpressed in many tumor types, our data indicate that targeting the SIX1–EYA complex may be a potent approach to inhibit tumor progression in multiple cancer types.

  19. Online Nanoflow RP-RP-MS Reveals Dynamics of Multi-component Ku Complex in Response to DNA Damage

    PubMed Central

    Zhou, Feng; Cardoza, Job D.; Ficarro, Scott B.; Adelmant, Guillaume O.; Lazaro, Jean-Bernard; Marto, Jarrod A.

    2010-01-01

    Tandem affinity purification (TAP) coupled with mass spectrometry has become the technique of choice for characterization of multi-component protein complexes. While current TAP protocols routinely provide high yield and specificity for proteins expressed under physiologically relevant conditions, analytical figures of merit required for efficient and in-depth LC-MS analysis remain unresolved. Here we implement a multidimensional chromatography platform, based on two stages of reversed-phase separation operated at high and low pH, respectively. We compare performance metrics for RP-RP and SCX-RP for the analysis of complex peptide mixtures derived from cell lysate, as well as protein complexes purified via TAP. Our data reveal that RP-RP fractionation outperforms SCX-RP primarily due to increased peak capacity in the first dimension separation. We integrate this system with miniaturized LC assemblies to achieve true online fractionation at low (≤5nL/min) effluent flow rates. Stable isotope labeling is used to monitor the dynamics of the multi-component Ku protein complex in response to DNA damage induced by gamma radiation. PMID:20873769

  20. Integrative genetic, epigenetic and pathological analysis of paraganglioma reveals complex dysregulation of NOTCH signaling.

    PubMed

    Cama, Alessandro; Verginelli, Fabio; Lotti, Lavinia Vittoria; Napolitano, Francesco; Morgano, Annalisa; D'Orazio, Andria; Vacca, Michele; Perconti, Silvia; Pepe, Felice; Romani, Federico; Vitullo, Francesca; di Lella, Filippo; Visone, Rosa; Mannelli, Massimo; Neumann, Hartmut P H; Raiconi, Giancarlo; Paties, Carlo; Moschetta, Antonio; Tagliaferri, Roberto; Veronese, Angelo; Sanna, Mario; Mariani-Costantini, Renato

    2013-10-01

    reveal an essential role of NOTCH pathway deregulation in this tumor type. PMID:23955600

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

    NASA Astrophysics Data System (ADS)

    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 < 10°) distances. Three component earthquake data is obtained from broadband seismic stations of Kandilli Observatory and Earthquake Research Center (KOERI, Turkey), Hellenic Unified Seismic Network (HUSN, Greece) and Earthquake Research Center of Turkey (AFAD-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

  2. Multiple mating reveals complex patterns of assortative mating by personality and body size.

    PubMed

    Montiglio, Pierre-Olivier; Wey, Tina W; Chang, Ann T; Fogarty, Sean; Sih, Andrew

    2016-01-01

    Understanding patterns of non-random mating is central to predicting the consequences of sexual selection. Most studies quantifying assortative mating focus on testing for correlations among partners' phenotypes in mated pairs. Few studies have distinguished between assortative mating arising from preferences for similar partners (expressed by all or a subset of the population) vs. from phenotypic segregation in the environment. Also, few studies have assessed the robustness of assortative mating against temporal changes in social conditions. We tracked multiple matings by stream water striders (Aquarius remigis) across variable social conditions to investigate mating patterns by both body size and behavioural type (personality). We documented temporal changes in partner availability and used a mixed model approach to analyse individual behaviours and changes in mating status recorded on an hourly basis. We assessed whether all or only a subset of individuals in the population expressed a tendency to mate with similar phenotypes. Our analyses took into account variation in the level of competition and in the phenotypes of available partners. Males and females exhibited significant assortative mating by body size: the largest males and females, and the smallest males and females mated together more often than random. However, individuals of intermediate size were equally likely to mate with small, intermediate or large partners. Individuals also displayed two contrasting patterns of assortative mating by personality (activity level). Individuals generally mated preferentially with partners of similar activity level. However, beyond that general trend, individuals with more extreme personalities tended to exhibit disassortative mating: the most active males mated disproportionately with less active females and the least active males tended to mate with more active females. Our analyses thus revealed multiple, distinct patterns of nonrandom mating. These mating

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

    PubMed

    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. PMID:27209795

  4. Complex frictional behaviour of clay-bearing carbonate faults revealed by integrated field and experimental investigation

    NASA Astrophysics Data System (ADS)

    Bullock, R. J.; De Paola, N.; Holdsworth, R.

    2013-12-01

    Seismicity in the Northern Apennines of Italy nucleates in and propagates through a complex carbonate multilayer sequence, comprising limestones with regular marl interbeds. Observations from the Gubbio fault (1984, Ms = 5.2) indicate that earthquake displacement is localized within narrow principal slip zones, <1.5 mm wide, characterized by cataclasites, gouges and calcite veins, and containing up to 50% phyllosilicate. Due to their predominantly velocity-strengthening nature, phyllosilicates are often considered to promote aseismic behaviour during the inter-seismic and post-seismic (afterslip) periods, and may act as barriers to rupture propagation in the upper crust. To assess the effect of clay content on the frictional behaviour and microstructural evolution of carbonate faults during earthquake propagation, we performed high-velocity friction (HVF) experiments, using a rotary-shear apparatus, on end-member gouges of calcite (Cal), montmorillonite (Mont) and mixed-layer illite-smectite (I/S), and on 50:50 and 80:20 mixtures of Cal+Mont and Cal+I/S. Experiments were conducted at room temperature, 1.3 m/s slip rate and 9 MPa normal load. Each sample was run under dry and water-saturated conditions, and experiments terminated both at peak friction (μp) and steady-state friction (μss) for comparison of microstructures at contrasting strength stages. Results for the dry gouges are in-line with previous HVF experiments: all samples attain a peak in friction at the onset of slip, ranging from 0.59 (Mont) to 0.72 (Cal), followed by a dramatic decrease in strength within the first ~0.5 m, after which friction maintains a constant μss value, ranging from 0.15 (Cal) to 0.22 (Mont). Frictional behaviour of the wet gouges is very different: clay-bearing samples typically do not exhibit a peak in friction; steady-state behaviour is attained immediately at the onset of sliding with friction coefficient values as low as 0.05. In summary, when dry, calcite controls the

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

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed Central

    Yuan, Bo; Liu, Pengfei; Gupta, Aditya; Beck, Christine R.; Tejomurtula, Anusha; Campbell, Ian M.; Gambin, Tomasz; Simmons, Alexandra D.; Withers, Marjorie A.; Harris, R. Alan; Rogers, Jeffrey; Schwartz, David C.; Lupski, James R.

    2015-01-01

    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. PMID:26641089

  7. Crystal structure of the S100A4-nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism.

    PubMed

    Kiss, Bence; Duelli, Annette; Radnai, László; Kékesi, Katalin A; Katona, Gergely; Nyitray, László

    2012-04-17

    S100A4 is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. It binds to the nonmuscle myosin IIA (NMIIA) tail near the assembly competence domain (ACD) promoting filament disassembly, which could be associated with increasing metastatic potential of tumor cells. Here, we investigate the mechanism of S100A4-NMIIA interaction based on binding studies and the crystal structure of S100A4 in complex with a 45-residue-long myosin heavy chain fragment. Interestingly, we also find that S100A4 binds as strongly to a homologous heavy chain fragment of nonmuscle myosin IIC as to NMIIA. The structure of the S100A4-NMIIA complex reveals a unique mode of interaction in the S100 family: A single, predominantly α-helical myosin chain is wrapped around the Ca(2+)-bound S100A4 dimer occupying both hydrophobic binding pockets. Thermal denaturation experiments of coiled-coil forming NMIIA fragments indicate that the coiled-coil partially unwinds upon S100A4 binding. Based on these results, we propose a model for NMIIA filament disassembly: Part of the random coil tailpiece and the C-terminal residues of the coiled-coil are wrapped around an S100A4 dimer disrupting the ACD and resulting in filament dissociation. The description of the complex will facilitate the design of specific drugs that interfere with the S100A4-NMIIA interaction. PMID:22460785

  8. The Structure of the BfrB-Bfd Complex Reveals Protein-Protein Interactions Enabling Iron Release from Bacterioferritin

    SciTech Connect

    Yao, Huili; Wang, Yan; Lovell, Scott; Kumar, Ritesh; Ruvinsky, Anatoly M; Battaile, Kevin P; Vakser, Ilya A; Rivera, Mario

    2012-09-11

    Ferritin-like molecules are unique to cellular iron homeostasis because they can store iron at concentrations much higher than those dictated by the solubility of Fe3+. Very little is known about the protein interactions that deliver iron for storage or promote the mobilization of stored iron from ferritin-like molecules. Here, we report the X-ray crystal structure of Pseudomonas aeruginosa bacterioferritin (Pa-BfrB) in complex with bacterioferritin-associated ferredoxin (Pa-Bfd) at 2.0 Å resolution. As the first example of a ferritin-like molecule in complex with a cognate partner, the structure provides unprecedented insight into the complementary interface that enables the [2Fe-2S] cluster of Pa-Bfd to promote heme-mediated electron transfer through the BfrB protein dielectric (~18 Å), a process that is necessary to reduce the core ferric mineral and facilitate mobilization of Fe2+. The Pa-BfrB-Bfd complex also revealed the first structure of a Bfd, thus providing a first view to what appears to be a versatile metal binding domain ubiquitous to the large Fer2_BFD family of proteins and enzymes with diverse functions. Residues at the Pa-BfrB-Bfd interface are highly conserved in Bfr and Bfd sequences from a number of pathogenic bacteria, suggesting that the specific recognition between Pa-BfrB and Pa-Bfd is of widespread significance to the understanding of bacterial iron homeostasis.

  9. Population structure of hyperinvasive serotype 12F, clonal complex 218 Streptococcus pneumoniae revealed by multilocus boxB sequence typing

    PubMed Central

    Rakov, Alexey V.; Ubukata, Kimiko; Robinson, D. Ashley

    2011-01-01

    At least four outbreaks of invasive disease caused by serotype 12F, clonal complex 218 Streptococcus pneumoniae have occurred in the United States over the past two decades. We studied the population structure of this clonal complex using a sample of 203 outbreak and surveillance isolates that were collected over 22 years from 34 US states and eight other countries. Conventional multilocus sequence typing identified five types and distinguished a single outbreak from the others. To improve typing resolution, multilocus boxB sequence typing (MLBT) was developed from 10 variable boxB minisatellite loci. MLBT identified 86 types and distinguished between each of the four outbreaks. Diversity across boxB loci tended to be positively correlated with repeat array size and, overall, best fit the infinite alleles mutation model. Multilocus linkage disequilibrium was strong, but pairwise disequilibrium decreased with the physical distance between loci and was strongest in one large region of the chromosome, indicating recent recombinations. Two major clusters were identified in the sample, and they were differentiated geographically, as western and more easterly US clusters, and temporally, as clusters that predominated before and after the licensure of pneumococcal conjugate vaccines. The diversity and linkage disequilibrium within these two clusters also differed, suggesting different population dynamics. MLBT revealed hidden aspects of the population structure of these hyperinvasive pneumococci, and it may provide a useful adjunct tool for outbreak investigations, surveillance, and population genetics studies of other pneumococcal clonal complexes. PMID:21888992

  10. Mechanism of Bacterial Cell-Surface Attachment Revealed by the Structure of Cellulosomal Type II Cohesin-dockerin Complex

    SciTech Connect

    Adams,J.; Pal, G.; Jia, Z.; Smith, S.

    2006-01-01

    Bacterial cell-surface attachment of macromolecular complexes maintains the microorganism in close proximity to extracellular substrates and allows for optimal uptake of hydrolytic byproducts. The cellulosome is a large multienzyme complex used by many anaerobic bacteria for the efficient degradation of plant cell-wall polysaccharides. The mechanism of cellulosome retention to the bacterial cell surface involves a calcium-mediated protein-protein interaction between the dockerin (Doc) module from the cellulosomal scaffold and a cohesin (Coh) module of cell-surface proteins located within the proteoglycan layer. Here, we report the structure of an ultra-high-affinity (K{sub a} = 1.44 x 10{sup 10} M{sup 1-}) complex between type II Doc, together with its neighboring X module from the cellulosome scaffold of Clostridium thermocellum, and a type II Coh module associated with the bacterial cell surface. Identification of X module-Doc and X module-Coh contacts reveal roles for the X module in Doc stability and enhanced Coh recognition. This extremely tight interaction involves one face of the Coh and both helices of the Doc and comprises significant hydrophobic character and a complementary extensive hydrogen-bond network. This structure represents a unique mechanism for cell-surface attachment in anaerobic bacteria and provides a rationale for discriminating between type I and type II Coh modules.

  11. The structure of the BfrB-Bfd complex reveals protein-protein interactions enabling iron release from bacterioferritin

    PubMed Central

    Yao, Huili; Wang, Yan; Lovell, Scott; Kumar, Ritesh; Ruvinsky, Anatoly M.; Battaile, Kevin P.; Vakser, Ilya A.; Rivera, Mario

    2012-01-01

    Ferritin-like molecules are unique to cellular iron homeostasis because they can store iron at concentrations much higher than those dictated by the solubility of Fe3+. Very little is known about the protein interactions that deliver iron for storage, or promote the mobilization of stored iron from ferritin-like molecules. Here, we report the X-ray crystal structure of Pseudomonas aeruginosa bacterioferritin (Pa-BfrB) in complex with bacterioferritin-associated ferredoxin (Pa-Bfd) at 2.0 Å resolution. As the first example of a ferritin-like molecule in complex with a cognate partner, the structure provides unprecedented insight into the complementary interface that enables the [2Fe-2S] cluster of Pa-Bfd to promote heme-mediated electron transfer through the BfrB protein dielectric (~18 Å), a process that is necessary to reduce the core ferric mineral and facilitate mobilization of Fe2+. The Pa-BfrB-Bfd complex also revealed the first structure of a Bfd, thus providing a first view to what appears to be a versatile metal binding domain ubiquitous to the large Fer2_BFD family of proteins and enzymes with diverse functions. Residues at the Pa-BfrB-Bfd interface are highly conserved in Bfr and Bfd sequences from a number of pathogenic bacteria, suggesting that the specific recognition between Pa-BfrB and Pa-Bfd is of widespread significance to the understanding of bacterial iron homeostasis. PMID:22812654

  12. Frequency and Factors Associated with Unexpected Death in an Acute Palliative Care Unit: Expect the Unexpected

    PubMed Central

    Bruera, Sebastian; Chisholm, Gary; Santos, Renata Dos; Bruera, Eduardo; Hui, David

    2015-01-01

    Context Few studies have examined the frequency of unexpected death and its associated factors in a palliative care setting. Objectives To determine the frequency of unexpected death in two acute palliative care units (APCUs); to compare the frequency of signs of impending death between expected and unexpected deaths; and to determine the predictors associated with unexpected death. Methods In this prospective, longitudinal, observational study, consecutive patients admitted to two APCUs were enrolled and physical signs of impending death were documented twice daily until discharge or death. Physicians were asked to complete a survey within 24 hours of APCU death. The death was considered unexpected if the physician answered “yes” to the question “Were you surprised by the timing of the death?” Results In total, 193 of 203 after-death assessments (95%) were collected for analysis. Nineteen of 193 patients died unexpectedly (10%). Signs of impending death, including nonreactive pupils, inability to close eyelids, decreased response to verbal stimuli, drooping of nasolabial folds, peripheral cyanosis, pulselessness of the radial artery, and respiration with mandibular movement, were documented more frequently in expected deaths than unexpected deaths (P < 0.05). Longer disease duration was associated with unexpected death (33 months vs. 12 months, P=0.009). Conclusion Unexpected death occurred in an unexpectedly high proportion of patients in the APCU setting, and was associated with fewer signs of impending death. Our findings highlight the need for palliative care teams to be prepared for the unexpected. PMID:25499421

  13. A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression.

    PubMed

    Wilson, William C; Hornig-Do, Hue-Tran; Bruni, Francesco; Chang, Jeong Ho; Jourdain, Alexis A; Martinou, Jean-Claude; Falkenberg, Maria; Spåhr, Henrik; Larsson, Nils-Göran; Lewis, Richard J; Hewitt, Lorraine; Baslé, Arnaud; Cross, Harold E; Tong, Liang; Lebel, Robert R; Crosby, Andrew H; Chrzanowska-Lightowlers, Zofia M A; Lightowlers, Robert N

    2014-12-01

    The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes. PMID:25008111

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

    NASA Astrophysics Data System (ADS)

    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

  15. The structure of an AspRS–tRNAAsp complex reveals a tRNA-dependent control mechanism

    PubMed Central

    Moulinier, L.; Eiler, S.; Eriani, G.; Gangloff, J.; Thierry, J.-C.; Gabriel, K.; McClain, W.H.; Moras, D.

    2001-01-01

    The 2.6 Å resolution crystal structure of an inactive complex between yeast tRNAAsp and Escherichia coli aspartyl-tRNA synthetase reveals the molecular details of a tRNA-induced mechanism that controls the specificity of the reaction. The dimer is asymmetric, with only one of the two bound tRNAs entering the active site cleft of its subunit. However, the flipping loop, which controls the proper positioning of the amino acid substrate, acts as a lid and prevents the correct positioning of the terminal adenosine. The structure suggests that the acceptor stem regulates the loop movement through sugar phosphate backbone– protein interactions. Solution and cellular studies on mutant tRNAs confirm the crucial role of the tRNA three-dimensional structure versus a specific recognition of bases in the control mechanism. PMID:11566892

  16. Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics.

    PubMed

    Uter, Nathan T; Perona, John J

    2004-10-01

    Pre-steady-state kinetic studies of Escherichia coli glutaminyl-tRNA synthetase conclusively demonstrate the existence of long-distance pathways of communication through the protein-RNA complex. Measurements of aminoacyl-tRNA synthesis reveal a rapid burst of product formation followed by a slower linear increase corresponding to k(cat). Thus, a step after chemistry but before regeneration of active enzyme is rate-limiting for synthesis of Gln-tRNA(Gln). Single-turnover kinetics validates these observations, confirming that the rate of the chemical step for tRNA aminoacylation (k(chem)) exceeds the steady-state rate by nearly 10-fold. The concentration dependence of the single-turnover reaction further reveals that the glutamine K(d) is significantly higher than the steady-state K(m) value. The separation of binding from catalytic events by transient kinetics now allows precise interpretation of how alterations in tRNA structure affect the aminoacylation reaction. Mutation of U35 in the tRNA anticodon loop decreases k(chem) by 30-fold and weakens glutamine binding affinity by 20-fold, demonstrating that the active-site configuration depends on enzyme-tRNA contacts some 40 A distant. By contrast, mutation of the adjacent G36 has very small effects on k(chem) and K(d) for glutamine. Together with x-ray crystallographic data, these findings allow a comparative evaluation of alternative long-range signaling pathways and lay the groundwork for systematic exploration of how induced-fit conformational transitions may control substrate selection in this model enzyme-RNA complex. PMID:15452355

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

    NASA Astrophysics Data System (ADS)

    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

  18. Genome-wide association analyses reveal complex genetic architecture underlying natural variation for flowering time in canola.

    PubMed

    Raman, H; Raman, R; Coombes, N; Song, J; Prangnell, R; Bandaranayake, C; Tahira, R; Sundaramoorthi, V; Killian, A; Meng, J; Dennis, E S; Balasubramanian, S

    2016-06-01

    Optimum flowering time is the key to maximize canola production in order to meet global demand of vegetable oil, biodiesel and canola-meal. We reveal extensive variation in flowering time across diverse genotypes of canola under field, glasshouse and controlled environmental conditions. We conduct a genome-wide association study and identify 69 single nucleotide polymorphism (SNP) markers associated with flowering time, which are repeatedly detected across experiments. Several associated SNPs occur in clusters across the canola genome; seven of them were detected within 20 Kb regions of a priori candidate genes; FLOWERING LOCUS T, FRUITFUL, FLOWERING LOCUS C, CONSTANS, FRIGIDA, PHYTOCHROME B and an additional five SNPs were localized within 14 Kb of a previously identified quantitative trait loci for flowering time. Expression analyses showed that among FLC paralogs, BnFLC.A2 accounts for ~23% of natural variation in diverse accessions. Genome-wide association analysis for FLC expression levels mapped not only BnFLC.C2 but also other loci that contribute to variation in FLC expression. In addition to revealing the complex genetic architecture of flowering time variation, we demonstrate that the identified SNPs can be modelled to predict flowering time in diverse canola germplasm accurately and hence are suitable for genomic selection of adaptative traits in canola improvement programmes. PMID:26428711

  19. Architecture and Nucleotide-Dependent Conformational Changes of the Rvb1-Rvb2 AAA+ Complex Revealed by Cryoelectron Microscopy.

    PubMed

    Ewens, Caroline A; Su, Min; Zhao, Liang; Nano, Nardin; Houry, Walid A; Southworth, Daniel R

    2016-05-01

    Rvb1 and Rvb2 are essential AAA+ proteins that interact together during the assembly and activity of diverse macromolecules including chromatin remodelers INO80 and SWR-C, and ribonucleoprotein complexes including telomerase and snoRNPs. ATP hydrolysis by Rvb1/2 is required for function; however, the mechanism that drives substrate remodeling is unknown. Here we determined the architecture of the yeast Rvb1/2 dodecamer using cryoelectron microscopy and identify that the substrate-binding insertion domain undergoes conformational changes in response to nucleotide state. 2D and 3D classification defines the dodecamer flexibility, revealing distinct arrangements and the hexamer-hexamer interaction interface. Reconstructions of the apo, ATP, and ADP states identify that Rvb1/2 undergoes substantial conformational changes that include a twist in the insertion-domain position and a corresponding rotation of the AAA+ ring. These results reveal how the ATP hydrolysis cycle of the AAA+ domains directs insertion-domain movements that could provide mechanical force during remodeling or helicase activities. PMID:27112599

  20. ARM-Seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments

    PubMed Central

    Cozen, Aaron E.; Quartley, Erin; Holmes, Andrew D.; Robinson, Eva H.; Phizicky, Eric M.; Lowe, Todd M.

    2015-01-01

    High throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA Methylation sequencing (ARM-Seq) which uses pre-treatment with Escherichia coli AlkB to demethylate 1-methyladenosine, 3-methylcytidine, and 1-methylguanosine, all commonly found in transfer RNAs. Comparative methylation analysis using ARM-Seq provides the first detailed, transcriptome-scale map of these modifications, and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-Seq demonstrates that tRNA-derived small RNAs accurately recapitulate the m1A modification state for well-characterized yeast tRNAs, and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-Seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state, and may reveal new roles for tRNA-derived RNAs as biomarkers or signaling molecules. PMID:26237225

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

    PubMed

    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. PMID:27618313

  2. Membrane association of the PTEN tumor suppressor: Neutron scattering and MD simulations reveal the structure of protein-membranes complexes

    PubMed Central

    Nanda, Hirsh; Heinrich, Frank; Lösche, Mathias

    2014-01-01

    Neutron reflection (NR) from planar interfaces is an emerging technology that provides unique and otherwise inaccessible structural information on disordered molecular systems such as membrane proteins associated with fluid bilayers, thus addressing one of the remaining challenges of structural biology. Although intrinsically a low-resolution technique, using structural information from crystallography or NMR allows the construction of NR models that describe the architecture of protein-membrane complexes at high resolution. In addition, a combination of these methods with molecular dynamics (MD) simulations has the potential to reveal the dynamics of protein interactions with the bilayer in atomistic detail. We review recent advances in this area by discussing the application of these techniques to the complex formed by the PTEN phosphatase with the plasma membrane. These studies provide insights in the cellular regulation of PTEN, its interaction with PI(4,5)P2 in the inner plasma membrane and the pathway by which its substrate, PI(3,4,5)P3, accesses the PTEN catalytic site. PMID:25461777

  3. RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair.

    PubMed

    Lescale, Chloé; Abramowski, Vincent; Bedora-Faure, Marie; Murigneux, Valentine; Vera, Gabriella; Roth, David B; Revy, Patrick; de Villartay, Jean-Pierre; Deriano, Ludovic

    2016-01-01

    XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2(c/c) mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2(c/c) XLF(-/-) p53(-/-) mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc/pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly. PMID:26833222

  4. Cryo-EM of Mitotic Checkpoint Complex-Bound APC/C Reveals Reciprocal and Conformational Regulation of Ubiquitin Ligation.

    PubMed

    Yamaguchi, Masaya; VanderLinden, Ryan; Weissmann, Florian; Qiao, Renping; Dube, Prakash; Brown, Nicholas G; Haselbach, David; Zhang, Wei; Sidhu, Sachdev S; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A

    2016-08-18

    The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). APC/C(CDC20) and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/C(CDC20)-UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here electron microscopy reveals conformational variation of APC/C(CDC20)-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly and conformational variability of APC/C modulate UBE2C-catalyzed ubiquitination of MCC's CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/C(CDC20)-MCC modulate E2 activation and determine distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation. PMID:27522463

  5. Structure of an E3:E2~Ub complex reveals an allosteric mechanism shared among RING/U-box ligases

    PubMed Central

    Pruneda, Jonathan N.; Littlefield, Peter J.; Soss, Sarah E.; Nordquist, Kyle A.; Chazin, Walter J.; Brzovic, Peter S.; Klevit, Rachel E.

    2012-01-01

    Despite the widespread importance of RING/U-box E3 ubiquitin ligases in ubiquitin (Ub) signaling, the mechanism by which this class of enzymes facilitates Ub transfer remains enigmatic. Here we present a structural model for a RING/U-box E3:E2~Ub complex poised for Ub transfer. The model and additional analyses reveal that E3 binding biases dynamic E2~Ub ensembles toward closed conformations with enhanced reactivity for substrate lysines. We identify a key hydrogen bond between a highly conserved E3 sidechain and an E2 backbone carbonyl, observed in all structures of active RING/U-Box E3/E2 pairs, as the linchpin for allosteric activation of E2~Ub. The conformational biasing mechanism is generalizable across diverse E2s and RING/U-box E3s, but is not shared by HECT-type E3s. The results provide a structural model for a RING/U-box E3:E2~Ub ligase complex and identify the long sought-after source of allostery for RING/U-Box activation of E2~Ub conjugates. PMID:22885007

  6. RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair

    PubMed Central

    Lescale, Chloé; Abramowski, Vincent; Bedora-Faure, Marie; Murigneux, Valentine; Vera, Gabriella; Roth, David B.; Revy, Patrick; de Villartay, Jean-Pierre; Deriano, Ludovic

    2016-01-01

    XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2c/c mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2c/c XLF−/− p53−/− mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc/pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly. PMID:26833222

  7. Amish Lifestyle Brings Unexpected Benefit: Less Asthma

    MedlinePlus

    ... 160228.html Amish Lifestyle Brings Unexpected Benefit: Less Asthma Finding suggests exposing kids to lots of allergens, ... rest of the population -- much lower rates of asthma. "We found Amish children had extremely low levels ...

  8. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation

    PubMed Central

    Lechtenberg, Bernhard C.; Rajput, Akhil; Sanishvili, Ruslan; Dobaczewska, Małgorzata K.; Ware, Carl F.; Mace, Peter D.; Riedl, Stefan J.

    2015-01-01

    Ubiquitination is a central process affecting all facets of cellular signaling and function1. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate2,3. The RBR family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases4–6. The RBR family includes Parkin4 and HOIP, the central catalytic factor of the linear ubiquitin chain assembly complex (LUBAC)7. While structural insights into the RBR E3 ligases Parkin and HHARI in their overall autoinhibited forms are available8–13, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely enigmatic. Here we present the first structure of the fully active HOIP-RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP-RBR adopts a conformation markedly different from that of autoinhibited RBRs. HOIP-RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centers ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, surprisingly, three distinct helix–IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~Ub conjugate as well as an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP-RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases. PMID:26789245

  9. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation.

    PubMed

    Lechtenberg, Bernhard C; Rajput, Akhil; Sanishvili, Ruslan; Dobaczewska, Małgorzata K; Ware, Carl F; Mace, Peter D; Riedl, Stefan J

    2016-01-28

    Ubiquitination is a central process affecting all facets of cellular signalling and function. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate. The RING-between-RING (RBR) family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases. The RBR family includes Parkin and HOIP, the central catalytic factor of the LUBAC (linear ubiquitin chain assembly complex). While structural insights into the RBR E3 ligases Parkin and HHARI in their overall auto-inhibited forms are available, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely unknown. Here we present the first structure, to our knowledge, of the fully active human HOIP RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP RBR adopts a conformation markedly different from that of auto-inhibited RBRs. HOIP RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centres ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, three distinct helix-IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~ubiquitin conjugate and, surprisingly, an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases. PMID:26789245

  10. Missed appendicitis: did unexpected intraluminal densities play a role?

    PubMed

    Harper, Rachel; Friedman, Benjamin T; Strote, Jared

    2016-01-01

    A healthy 19-year-old boy presented to our emergency department with abdominal pain. His history, examination and laboratory evaluation raised concern for appendicitis. A CT study of the abdomen and pelvis was carried out by the radiologist and emergency physician and was notable only for a large amount of unexpected high-attenuation intraluminal material. With further history, this was thought to be most likely retained bismuth from over-the-counter medicine ingestion. The patient was discharged home without a diagnosis. Further review of the CT scan by a second radiologist revealed a concern for appendiceal enlargement and associated free fluid. The patient was called back for further evaluation and treatment and ultimately an appendectomy was performed. Physicians should be aware of the causes and impact of unexpected radiopaque intraluminal contents on radiological studies. Most commonly from ingested medicine, such findings can obscure mucosal details, mimic active bleeding or create a distraction from other abnormalities. PMID:27605197

  11. Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation

    PubMed Central

    Guo, Yu; Wang, Wenming; Sun, Yuna; Ma, Chao; Wang, Xu; Wang, Xin; Liu, Pi; Shen, Shu; Li, Baobin; Lin, Jianping; Deng, Fei

    2015-01-01

    ABSTRACT Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcore exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. IMPORTANCE Hantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and

  12. Initial rate kinetic studies show an unexpected influence of para-substituents on the catalytic behaviour of manganese complexes of TMTACN in the epoxidation of styrenes with H2O2.

    PubMed

    Ilyashenko, Gennadiy; De Faveri, Giorgio; Masoudi, Shirin; Al-Safadi, Rawan; Watkinson, Michael

    2013-03-28

    Investigations into the efficacy of a range of enantiomerically pure BINOL additives in the epoxidation of styrene substrates with a number of manganese catalysts prepared from the ligand 1,4,7-trimethyl-1,4,7-triazacyclononane, TMTACN, using hydrogen peroxide as the oxidant have revealed that there are fundamental differences in reactivity between apparently similar systems. Whilst no asymmetric induction was obtained in the styrene oxide products formed, the data obtained from initial rate kinetic studies appear to be consistent with a number of different catalytically active species operating, the nature of which are profoundly affected by the starting materials used. PMID:23358659

  13. Co-modulation analysis of gene regulation in breast cancer reveals complex interplay between ESR1 and ERBB2 genes

    PubMed Central

    2015-01-01

    Background Gene regulation is dynamic across cellular conditions and disease subtypes. From the aspect of regulation under modulation, regulation strength between a pair of genes can be modulated by (dependent on) expression abundance of another gene (modulator gene). Previous studies have demonstrated the involvement of genes modulated by single modulator genes in cancers, including breast cancer. However, analysis of multi-modulator co-modulation that can further delineate the landscape of complex gene regulation is, to our knowledge, unexplored previously. In the present study we aim to explore the joint effects of multiple modulator genes in modulating global gene regulation and dissect the biological functions in breast cancer. Results To carry out the analysis, we proposed the Covariability-based Multiple Regression (CoMRe) method. The method is mainly built on a multiple regression model that takes expression levels of multiple modulators as inputs and regulation strength between genes as output. Pairs of genes were divided into groups based on their co-modulation patterns. Analyzing gene expression profiles from 286 breast cancer patients, CoMRe investigated ten candidate modulator genes that interacted and jointly determined global gene regulation. Among the candidate modulators, ESR1, ERBB2, and ADAM12 were found modulating the most numbers of gene pairs. The largest group of gene pairs was composed of ones that were modulated by merely ESR1. Functional annotation revealed that the group was significantly related to tumorigenesis and estrogen signaling in breast cancer. ESR1−ERBB2 co-modulation was the largest group modulated by more than one modulators. Similarly, the group was functionally associated with hormone stimulus, suggesting that functions of the two modulators are performed, at least partially, through modulation. The findings were validated in majorities of patients (> 99%) of two independent breast cancer datasets. Conclusions We have

  14. Complex patterns of divergence among green-sensitive (RH2a) African cichlid opsins revealed by Clade model analyses

    PubMed Central

    2012-01-01

    Background Gene duplications play an important role in the evolution of functional protein diversity. Some models of duplicate gene evolution predict complex forms of paralog divergence; orthologous proteins may diverge as well, further complicating patterns of divergence among and within gene families. Consequently, studying the link between protein sequence evolution and duplication requires the use of flexible substitution models that can accommodate multiple shifts in selection across a phylogeny. Here, we employed a variety of codon substitution models, primarily Clade models, to explore how selective constraint evolved following the duplication of a green-sensitive (RH2a) visual pigment protein (opsin) in African cichlids. Past studies have linked opsin divergence to ecological and sexual divergence within the African cichlid adaptive radiation. Furthermore, biochemical and regulatory differences between the RH2aα and RH2aβ paralogs have been documented. It thus seems likely that selection varies in complex ways throughout this gene family. Results Clade model analysis of African cichlid RH2a opsins revealed a large increase in the nonsynonymous-to-synonymous substitution rate ratio (ω) following the duplication, as well as an even larger increase, one consistent with positive selection, for Lake Tanganyikan cichlid RH2aβ opsins. Analysis using the popular Branch-site models, by contrast, revealed no such alteration of constraint. Several amino acid sites known to influence spectral and non-spectral aspects of opsin biochemistry were found to be evolving divergently, suggesting that orthologous RH2a opsins may vary in terms of spectral sensitivity and response kinetics. Divergence appears to be occurring despite intronic gene conversion among the tandemly-arranged duplicates. Conclusions Our findings indicate that variation in selective constraint is associated with both gene duplication and divergence among orthologs in African cichlid RH2a opsins. At

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

    SciTech Connect

    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.

  16. Ancient DNA Reveals Prehistoric Gene-Flow from Siberia in the Complex Human Population History of North East Europe

    PubMed Central

    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

  17. Structure of Severe Fever with Thrombocytopenia Syndrome Virus Nucleocapsid Protein in Complex with Suramin Reveals Therapeutic Potential

    PubMed Central

    Jiao, Lianying; Ouyang, Songying; Liang, Mifang; Niu, Fengfeng; Shaw, Neil; Wu, Wei; Ding, Wei; Jin, Cong; Peng, Yao; Zhu, Yanping; Zhang, Fushun; Wang, Tao; Li, Chuan; Zuo, Xiaobing; Luan, Chi-Hao; Li, Dexin

    2013-01-01

    Severe fever with thrombocytopenia syndrome is an emerging infectious disease caused by a novel bunyavirus (SFTSV). Lack of vaccines and inadequate therapeutic treatments have made the spread of the virus a global concern. Viral nucleocapsid protein (N) is essential for its transcription and replication. Here, we present the crystal structures of N from SFTSV and its homologs from Buenaventura (BUE) and Granada (GRA) viruses. The structures reveal that phleboviral N folds into a compact core domain and an extended N-terminal arm that mediates oligomerization, such as tetramer, pentamer, and hexamer of N assemblies. Structural superimposition indicates that phleboviral N adopts a conserved architecture and uses a similar RNA encapsidation strategy as that of RVFV-N. The RNA binding cavity runs along the inner edge of the ring-like assembly. A triple mutant of SFTSV-N, R64D/K67D/K74D, almost lost its ability to bind RNA in vitro, is deficient in its ability to transcribe and replicate. Structural studies of the mutant reveal that both alterations in quaternary assembly and the charge distribution contribute to the loss of RNA binding. In the screening of inhibitors Suramin was identified to bind phleboviral N specifically. The complex crystal structure of SFTSV-N with Suramin was refined to a 2.30-Å resolution. Suramin was found sitting in the putative RNA binding cavity of SFTSV-N. The inhibitory effect of Suramin on SFTSV replication was confirmed in Vero cells. Therefore, a common Suramin-based therapeutic approach targeting SFTSV-N and its homologs could be developed for containing phleboviral outbreaks. PMID:23576501

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

    PubMed Central

    Bauerfeind, Martin Andreas; Winkelmann, Traud; Franken, Philipp; Druege, Uwe

    2015-01-01

    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 4 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 3 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 tolerant cultivars

  19. Whole Genome Sequencing Reveals Complex Evolution Patterns of Multidrug-Resistant Mycobacterium tuberculosis Beijing Strains in Patients

    PubMed Central

    Merker, Matthias; Kohl, Thomas A.; Roetzer, Andreas; Truebe, Leona; Richter, Elvira; Rüsch-Gerdes, Sabine; Fattorini, Lanfranco; Oggioni, Marco R.; Cox, Helen; Varaine, Francis; Niemann, Stefan

    2013-01-01

    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 molecular drug

  20. RNA Profiles of Porcine Embryos during Genome Activation Reveal Complex Metabolic Switch Sensitive to In Vitro Conditions

    PubMed Central

    Østrup, Olga; Olbricht, Gayla; Østrup, Esben; Hyttel, Poul; Collas, Philippe; Cabot, Ryan

    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, 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 human embryos

  1. Three-Dimensional Analysis of a Viral RNA Replication Complex Reveals a Virus-Induced Mini-Organelle

    PubMed Central

    Kopek, Benjamin G; Perkins, Guy; Miller, David J; Ellisman, Mark H; Ahlquist, Paul

    2007-01-01

    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 ∼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 ∼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 ∼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. PMID:17696647

  2. Investigating Initial Disclosures and Reactions to Unexpected, Positive HPV Diagnosis

    PubMed Central

    Smith, Rachel A.; Hernandez, Rachael; Catona, Danielle

    2013-01-01

    Initial disclosures of health conditions are critical communication moments. Existing research focuses on disclosers; integrating confidants into studies of initial disclosures is needed. Guided by the disclosure decision-making model (DD-MM; Greene, 2009), this study examined what diagnosed persons and confidants may say when faced with unexpected test results and unexpected disclosures, respectively. Participants (N = 151) recorded an audio-visual message for another person, after imagining that they or the other person had just received unexpected, positive HPV test results. The qualitative analysis revealed four themes: (1) impression management and social distance, (2) invisible symptoms and advice regarding future disclosures, (3) expressing and acknowledging emotional reactions, and (4) misunderstandings and lacking knowledge about HPV. These findings suggested that DD-MM may be a relevant framework for understanding not only when disclosers share, but what disclosers and confidants say in early conversations about new diagnoses. While disclosers’ and confidants’ messages showed marked similarities, important differences appeared. For example, confidants focused on assuaging disclosers’ fear about the consequences, whereas disclosers expressed distress related to their uncertainty about the prognosis of an HPV infection and how to prepare for next steps. The discussion highlighted implications for the DD-MM, HPV disclosures, and future interventions. PMID:25642121

  3. A Complex Regulatory Network Coordinating Cell Cycles During C. elegans Development Is Revealed by a Genome-Wide RNAi Screen

    PubMed Central

    Roy, Sarah H.; Tobin, David V.; Memar, Nadin; Beltz, Eleanor; Holmen, Jenna; Clayton, Joseph E.; Chiu, Daniel J.; Young, Laura D.; Green, Travis H.; Lubin, Isabella; Liu, Yuying; Conradt, Barbara; Saito, R. Mako

    2014-01-01

    The development and homeostasis of multicellular animals requires precise coordination of cell division and differentiation. We performed a genome-wide RNA interference screen in Caenorhabditis elegans to reveal the components of a regulatory network that promotes developmentally programmed cell-cycle quiescence. The 107 identified genes are predicted to constitute regulatory networks that are conserved among higher animals because almost half of the genes are represented by clear human orthologs. Using a series of mutant backgrounds to assess their genetic activities, the RNA interference clones displaying similar properties were clustered to establish potential regulatory relationships within the network. This approach uncovered four distinct genetic pathways controlling cell-cycle entry during intestinal organogenesis. The enhanced phenotypes observed for animals carrying compound mutations attest to the collaboration between distinct mechanisms to ensure strict developmental regulation of cell cycles. Moreover, we characterized ubc-25, a gene encoding an E2 ubiquitin-conjugating enzyme whose human ortholog, UBE2Q2, is deregulated in several cancers. Our genetic analyses suggested that ubc-25 acts in a linear pathway with cul-1/Cul1, in parallel to pathways employing cki-1/p27 and lin-35/pRb to promote cell-cycle quiescence. Further investigation of the potential regulatory mechanism demonstrated that ubc-25 activity negatively regulates CYE-1/cyclin E protein abundance in vivo. Together, our results show that the ubc-25-mediated pathway acts within a complex network that integrates the actions of multiple molecular mechanisms to control cell cycles during development. PMID:24584095

  4. Complex chemoattractive and chemorepellent Kit signals revealed by direct imaging of murine mast cells in microfluidic gradient chambers†

    PubMed Central

    Shamloo, Amir; Manchandia, Milan; Ferreira, Meghaan; Mani, Maheswaran; Nguyen, Christopher; Jahn, Thomas; Weinberg, Kenneth

    2014-01-01

    Besides its cooperating effects on stem cell proliferation and survival, Kit ligand (KL) is a potent chemotactic protein. While transwell assays permit studies of the frequency of migrating cells, the lack of direct visualization precludes dynamic chemotaxis studies. In response, we utilize microfluidic chambers that enable direct observation of murine bone marrow-derived mast cells (BMMC) within stable KL gradients. Using this system, individual Kit+ BMMC were quantitatively analyzed for migration speed and directionality during KL-induced chemotaxis. Our results indicated a minimum activating threshold of ~3 ng ml−1 for chemoattraction. Analysis of cells at KL concentrations below 3 ng ml−1 revealed a paradoxical chemorepulsion, which has not been described previously. Unlike chemoattraction, which occurred continuously after an initial time lag, chemorepulsion occurred only during the first 90 minutes of observation. Both chemoattraction and chemorepulsion required the action of G-protein coupled receptors (GPCR), as treatment with pertussis toxin abrogated directed migration. These results differ from previous studies of GPCR-mediated chemotaxis, where chemorepulsion occurred at high ligand concentrations. These data indicate that Kit-mediated chemotaxis is more complex than previously understood, with the involvement of GPCRs in addition to the Kit receptor tyrosine kinase and the presence of both chemoattractive and chemorepellent phases. PMID:23835699

  5. Genome Re-Sequencing of Semi-Wild Soybean Reveals a Complex Soja Population Structure and Deep Introgression

    PubMed Central

    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. PMID:25265539

  6. Structure and dynamics of photosystem II light-harvesting complex revealed by high-resolution FTICR mass spectrometric proteome analysis.

    PubMed

    Galetskiy, Dmitry; Susnea, Iuliana; Reiser, Verena; Adamska, Iwona; Przybylski, Michael

    2008-07-01

    Structure and dynamics of membrane-bound light-harvesting pigment-protein complexes (LHCs), which collect and transmit light energy for photosynthesis and thereby play an essential role in the regulation of photosynthesis and photoprotection, were identified and characterized using high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). LHCs from photosystem II (LHCII) were isolated from the thylakoid membrane of Arabidopsis thaliana leaves after light stress treatment using sucrose density gradient centrifugation, and separated by gel-filtration into LHCII subcomplexes. Using reversed-phase high-performance liquid chromatography and two-dimensional (2D) gel electrophoresis, the LHCII proteins, Lhcb1-6 and fibrillins, were efficiently separated and identified by FTICR-MS. Some of the LHCII subcomplexes were shown to migrate from photosystem II to photosystem I as a result of short-term adaptation to changes in light intensity. In the mobile LHCII subcomplexes, decreased levels of fibrillins and a modified composition of LHCII protein isoforms were identified compared to the tightly bound LHCII subcomplexes. In addition, FTICR-MS analysis revealed several oxidative modifications of LHCII proteins. A number of protein spots in 2D gels were found to contain a mixture of proteins, illustrating the feasibility of high-resolution mass spectrometry to identify proteins that remain unseparated in 2D gels even upon extended pH gradients. PMID:18455927

  7. Prefrontal cortical recordings with biomorphic MEAs reveal complex columnar-laminar microcircuits for BCI/BMI implementation

    PubMed Central

    Opris, Ioan; Fuqua, Joshua L; Gerhardt, Greg A.; Hampson, Robert E.; Deadwyler, Sam A.

    2015-01-01

    The mammalian prefrontal cortex known as the seat of high brain functions uses a six layer distribution of minicolumnar neurons to coordinate the integration of sensory information and the selection of relevant signals for goal driven behavior. To reveal the complex functionality of these columnar microcircuits we employed simultaneous recordings with several configurations of biomorphic microelectrode arrays (MEAs) within cortical layers in adjacent minicolumns, in four nohuman primates (NHPs) performing a delayed match-to-sample (DMS) visual discrimination task. We examined: 1) the functionality of inter-laminar, and inter-columnar interactions between pairs of cells in the same or different minicolumns by use of normalized cross-correlation histograms (CCH), 2) the modulation of Glutamate concentration in layer 2/3, and 3) the potential interactions within these microcircuits. The results demonstrate that neurons in both infra-granular and supra-granular layers interact through inter-laminar loops, as well as through intra-laminar to produce behavioral response signals. These results provide new insights into the manner in which prefrontal cortical microcircuitry integrates sensory stimuli used to provide behaviorally relevant signals that may be implemented in brain computer/machine interfaces (BCI/BMIs) during performance of the task. PMID:24954713

  8. Structure of trigger factor binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action

    SciTech Connect

    Baram, David; Pyetan, Erez; Sittner, Assa; Auerbach-Nevo, Tamar; Bashan, Anat; Yonath, Ada

    2010-07-13

    Trigger factor (TF), the first chaperone in eubacteria to encounter the emerging nascent chain, binds to the large ribosomal subunit in the vicinity of the protein exit tunnel opening and forms a sheltered folding space. Here, we present the 3.5-{angstrom} crystal structure of the physiological complex of the large ribosomal subunit from the eubacterium Deinococcus radiodurans with the N-terminal domain of TF (TFa) from the same organism. For anchoring, TFa exploits a small ribosomal surface area in the vicinity of proteins L23 and L29, by using its 'signature motif' as well as additional structural elements. The molecular details of TFa interactions reveal that L23 is essential for the association of TF with the ribosome and may serve as a channel of communication with the nascent chain progressing in the tunnel. L29 appears to induce a conformational change in TFa, which results in the exposure of TFa hydrophobic patches to the opening of the ribosomal exit tunnel, thus increasing its affinity for hydrophobic segments of the emerging nascent polypeptide. This observation implies that, in addition to creating a protected folding space for the emerging nascent chain, TF association with the ribosome prevents aggregation by providing a competing hydrophobic environment and may be critical for attaining the functional conformation necessary for chaperone activity.

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

    PubMed

    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. PMID:24660979

  10. Virtual screening reveals a viral-like polymerase inhibitor that complexes with the DNA polymerase of Moniliophthora perniciosa.

    PubMed

    Andrade, B S; Souza, C S; Santos, G; Góes-Neto, A

    2016-01-01

    The filamentous fungus Moniliophthora perniciosa is a basidiomycota that causes the witches' broom disease in cocoa trees (Theobroma cacao L.). The mitochondrial DNA polymerase of M. perniciosa (MpmitDNApol) is classified within the B family of DNA polymerases, which can be found in viruses and cellular organelles. Using virtual screening processes, accessing KEGG, PubChem, and ZINC databases, we selected the 27 best putative nucleoside viral-like polymerase inhibitors to test against MpmitDNApol. We used Autodock Vina to perform docking simulations of the selected molecules and to return energy values in several ligand conformations. Then, we used Pymol v1.7.4.4 to check the stereochemistry of chiral carbons, hydrogen bonding receptors, absence or presence of hydrogen, sub and superstructure, numbers of rings, rotatable bonds, and donor groups. We selected the Entecavir Hydrate, a drug used to control hepatitis B; subsequently AMBER 14 was used to describe the behavior of polymerase-entecavir complex after setting up 3500 ps of simulation in water at a temperature of 300 K. From the simulation, a graph of Potential Energy was generated revealing that the ligand remains in the catalytic site after 3500 ps with a final energy of -612,587.4214 kcal/mol. PMID:27323084

  11. Crystal structure of PXY-TDIF complex reveals a conserved recognition mechanism among CLE peptide-receptor pairs

    PubMed Central

    Zhang, Heqiao; Lin, Xiaoya; Han, Zhifu; Qu, Li-Jia; Chai, Jijie

    2016-01-01

    Plants can achieve amazing lifespans because of their continuous and repetitive formation of new organs by stem cells present within meristems. The balance between proliferation and differentiation of meristem cells is largely regulated by the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) peptide hormones. One of the well-characterized CLE peptides, CLE41/TDIF (tracheary elements differentiation inhibitory factor), functions to suppress tracheary element differentiation and promote procambial cell proliferation, playing important roles in vascular development and wood formation. The recognition mechanisms of TDIF or other CLE peptides by their respective receptors, however, remain largely elusive. Here we report the crystal structure of TDIF in complex with its receptor PXY, a leucine-rich repeat receptor kinase (LRR-RK). Our structure reveals that TDIF mainly adopts an “Ω”-like conformation binding to the inner surface of the LRR domain of PXY. Interaction between TDIF and PXY is predominately mediated by the relatively conserved amino acids of TDIF. Structure-based sequence alignment showed that the TDIF-interacting motifs are also conserved among other known CLE receptors. Our data provide a structural template for understanding the recognition mechanism of CLE peptides by their receptors, offering an opportunity for the identification of receptors of other uncharacterized CLE peptides. PMID:27055373

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

    SciTech Connect

    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

    2008-04-29

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

    SciTech Connect

    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

    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.

  14. Cryo-EM of ribosomal 80S complexes with termination factors reveal the translocated cricket paralysis virus IRES

    PubMed Central

    Muhs, Margarita; Hilal, Tarek; Mielke, Thorsten; Skabkin, Maxim A.; Sanbonmatsu, Karissa Y.; Pestova, Tatyana V.; Spahn, Christian M.T.

    2015-01-01

    Summary The Cricket paralysis virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the CrPV IRES jump starts translation in the elongation phase from the ribosomal A-site. Here we present cryo-EM maps of 80S•CrPV-STOP•eRF1•eRF3•GMPPNP and 80S•CrPV-STOP•eRF1 complexes revealing a previously unseen binding state of the IRES and directly rationalizing that an eEF2-dependent translocation of the IRES is required to allow the first A-site occupation. During this unusual translocation event the IRES undergoes a pronounced conformational change to a more stretched conformation. At the same time our structural analysis provides information about the binding modes of eRF1•eRF3•GMPPNP and eRF1 in a minimal system. It shows that neither eRF3 nor ABCE1 are required for the active conformation of eRF1 at the intersection between eukaryotic termination and recycling. PMID:25601755

  15. Small RNA and Degradome Sequencing Reveal Complex Roles of miRNAs and Their Targets in Developing Wheat Grains

    PubMed Central

    Geng, Yuke; Hao, Chenyang; Chen, Xinhong; Zhang, Xueyong

    2015-01-01

    Plant microRNAs (miRNAs) have been shown to play critical roles in plant development. In this study, we employed small RNA combined with degradome sequencing to survey development-related miRNAs and their validated targets during wheat grain development. A total of 186 known miRNAs and 37 novel miRNAs were identified in four small RNA libraries. Moreover, a miRNA-like long hairpin locus was first identified to produce 21~22-nt phased siRNAs that act in trans to cleave target mRNAs. A comparison of the miRNAomes revealed that 55 miRNA families were differentially expressed during the grain development. Predicted and validated targets of these development-related miRNAs are involved in different cellular responses and metabolic processes including cell proliferation, auxin signaling, nutrient metabolism and gene expression. This study provides insight into the complex roles of miRNAs and their targets in regulating wheat grain development. PMID:26426440

  16. Gene Coexpression Analysis Reveals Complex Metabolism of the Monoterpene Alcohol Linalool in Arabidopsis Flowers[W][OPEN

    PubMed Central

    Ginglinger, Jean-François; Boachon, Benoit; Höfer, René; Paetz, Christian; Köllner, Tobias G.; Miesch, Laurence; Lugan, Raphael; Baltenweck, Raymonde; Mutterer, Jérôme; Ullmann, Pascaline; Beran, Franziska; Claudel, Patricia; Verstappen, Francel; Fischer, Marc J.C.; Karst, Francis; Bouwmeester, Harro; Miesch, Michel; Schneider, Bernd; Gershenzon, Jonathan; Ehlting, Jürgen; Werck-Reichhart, Danièle

    2013-01-01

    The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (−)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined. PMID:24285789

  17. Structural and Computational Studies of the Staphylococcus aureus Sortase B-Substrate Complex Reveal a Substrate-stabilized Oxyanion Hole*

    PubMed Central

    Jacobitz, Alex W.; Wereszczynski, Jeff; Yi, Sung Wook; Amer, Brendan R.; Huang, Grace L.; Nguyen, Angelyn V.; Sawaya, Michael R.; Jung, Michael E.; McCammon, J. Andrew; Clubb, Robert T.

    2014-01-01

    Sortase cysteine transpeptidases covalently attach proteins to the bacterial cell wall or assemble fiber-like pili that promote bacterial adhesion. Members of this enzyme superfamily are widely distributed in Gram-positive bacteria that frequently utilize multiple sortases to elaborate their peptidoglycan. Sortases catalyze transpeptidation using a conserved active site His-Cys-Arg triad that joins a sorting signal located at the C terminus of their protein substrate to an amino nucleophile located on the cell surface. However, despite extensive study, the catalytic mechanism and molecular basis of substrate recognition remains poorly understood. Here we report the crystal structure of the Staphylococcus aureus sortase B enzyme in a covalent complex with an analog of its NPQTN sorting signal substrate, revealing the structural basis through which it displays the IsdC protein involved in heme-iron scavenging from human hemoglobin. The results of computational modeling, molecular dynamics simulations, and targeted amino acid mutagenesis indicate that the backbone amide of Glu224 and the side chain of Arg233 form an oxyanion hole in sortase B that stabilizes high energy tetrahedral catalytic intermediates. Surprisingly, a highly conserved threonine residue within the bound sorting signal substrate facilitates construction of the oxyanion hole by stabilizing the position of the active site arginine residue via hydrogen bonding. Molecular dynamics simulations and primary sequence conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of enzymes within the sortase superfamily. PMID:24519933

  18. High-resolution imaging reveals new features of nuclear export of mRNA through the nuclear pore complexes.

    PubMed

    Kelich, Joseph M; Yang, Weidong

    2014-01-01

    The nuclear envelope (NE) of eukaryotic cells provides a physical barrier for messenger RNA (mRNA) and the associated proteins (mRNPs) traveling from sites of transcription in the nucleus to locations of translation processing in the cytoplasm. Nuclear pore complexes (NPCs) embedded in the NE serve as a dominant gateway for nuclear export of mRNA. However, the fundamental characterization of export dynamics of mRNPs through the NPC has been hindered by several technical limits. First, the size of NPC that is barely below the diffraction limit of conventional light microscopy requires a super-resolution microscopy imaging approach. Next, the fast transit of mRNPs through the NPC further demands a high temporal resolution by the imaging approach. Finally, the inherent three-dimensional (3D) movements of mRNPs through the NPC demand the method to provide a 3D mapping of both transport kinetics and transport pathways of mRNPs. This review will highlight the recently developed super-resolution imaging techniques advanced from 1D to 3D for nuclear export of mRNPs and summarize the new features in the dynamic nuclear export process of mRNPs revealed from these technical advances. PMID:25141104

  19. Comparative proteomic analysis revealing the complex network associated with waterlogging stress in maize (Zea mays L.) seedling root cells.

    PubMed

    Yu, Feng; Han, Xuesong; Geng, Cunjuan; Zhao, Yanxin; Zhang, Zuxin; Qiu, Fazhan

    2015-01-01

    Soil waterlogging is one of the major abiotic stresses affecting maize grain yields. To understand the molecular mechanisms underlying waterlogging tolerance in maize, the iTRAQ LC-MS/MS technique was employed to map the proteomes of seedling root cells of the A3237 (tolerant inbred) and A3239 (sensitive inbred) lines under control and waterlogging conditions. Among the 3318 proteins identified, 211 were differentially abundant proteins (DAPs), of which 81 were specific to A3237 and 57 were specific to A3239. These DAPs were categorized into 11 groups that were closely related to the plant stress response, including metabolism, energy, transport, and disease/defense. In the waterlogged A3237 root cells, NADP-malic enzyme, glutamate decarboxylase, coproporphyrinogen III oxidase, GSH S-transferase, GSH dehydrogenase, and xyloglucan endotransglycosylase 6 were specifically accumulated to manage energy consumption, maintain pH levels, and minimize oxidative damage. The evaluations of five specific physiological parameters (alcohol dehydrogenase activity and GSH, malondialdehyde, adenosine 5'-triphosphate, and nicotinamide adenine dinucleotide concentrations) were in agreement with the proteomic results. Moreover, based on the proteomic assay, eight representative genes encoding DAPs were selected for validation at the transcriptional level. qRT-PCR revealed that the expression levels of these genes correlated with their observed protein abundance. These findings shed light on the complex mechanisms underlying waterlogging tolerance in maize. All MS data have been deposited into the ProteomeXchange with the identifier PXD001125 http://proteomecentral.proteomexchange.org/dataset/PXD001125. PMID:25316036

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

    PubMed Central

    Hoppins, Suzanne; Collins, Sean R.; Cassidy-Stone, Ann; Hummel, Eric; DeVay, Rachel M.; Lackner, Laura L.; Westermann, Benedikt; Schuldiner, Maya

    2011-01-01

    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. PMID:21987634

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

    PubMed

    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. PMID:25265539

  2. Crystal structure of PXY-TDIF complex reveals a conserved recognition mechanism among CLE peptide-receptor pairs.

    PubMed

    Zhang, Heqiao; Lin, Xiaoya; Han, Zhifu; Qu, Li-Jia; Chai, Jijie

    2016-05-01

    Plants can achieve amazing lifespans because of their continuous and repetitive formation of new organs by stem cells present within meristems. The balance between proliferation and differentiation of meristem cells is largely regulated by the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) peptide hormones. One of the well-characterized CLE peptides, CLE41/TDIF (tracheary elements differentiation inhibitory factor), functions to suppress tracheary element differentiation and promote procambial cell proliferation, playing important roles in vascular development and wood formation. The recognition mechanisms of TDIF or other CLE peptides by their respective receptors, however, remain largely elusive. Here we report the crystal structure of TDIF in complex with its receptor PXY, a leucine-rich repeat receptor kinase (LRR-RK). Our structure reveals that TDIF mainly adopts an "Ω"-like conformation binding to the inner surface of the LRR domain of PXY. Interaction between TDIF and PXY is predominately mediated by the relatively conserved amino acids of TDIF. Structure-based sequence alignment showed that the TDIF-interacting motifs are also conserved among other known CLE receptors. Our data provide a structural template for understanding the recognition mechanism of CLE peptides by their receptors, offering an opportunity for the identification of receptors of other uncharacterized CLE peptides. PMID:27055373

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

    PubMed

    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. PMID:26116530

  4. Cryo-EM of ribosomal 80S complexes with termination factors reveals the translocated cricket paralysis virus IRES.

    PubMed

    Muhs, Margarita; Hilal, Tarek; Mielke, Thorsten; Skabkin, Maxim A; Sanbonmatsu, Karissa Y; Pestova, Tatyana V; Spahn, Christian M T

    2015-02-01

    The cricket paralysis virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the CrPV IRES jumpstarts translation in the elongation phase from the ribosomal A site. Here, we present cryoelectron microscopy (cryo-EM) maps of 80S⋅CrPV-STOP ⋅ eRF1 ⋅ eRF3 ⋅ GMPPNP and 80S⋅CrPV-STOP ⋅ eRF1 complexes, revealing a previously unseen binding state of the IRES and directly rationalizing that an eEF2-dependent translocation of the IRES is required to allow the first A-site occupation. During this unusual translocation event, the IRES undergoes a pronounced conformational change to a more stretched conformation. At the same time, our structural analysis provides information about the binding modes of eRF1 ⋅ eRF3 ⋅ GMPPNP and eRF1 in a minimal system. It shows that neither eRF3 nor ABCE1 are required for the active conformation of eRF1 at the intersection between eukaryotic termination and recycling. PMID:25601755

  5. High-density PhyloChip profiling of stimulated aquifer microbial communities reveals a complex response to acetate amendment

    SciTech Connect

    Handley, Kim M.; Wrighton, Kelly C.; Piceno, Yvette M.; Andersen, Gary L.; DeSantis, Todd Z.; Williams, Kenneth H.; Wilkins, Michael J.; N'Guessan, A. Lucie; Peacock, Aaron; Bargar, John; Long, Philip E.; Banfield, Jillian F.

    2012-04-13

    There is increasing interest in harnessing the functional capacities of indigenous microbial communities to transform and remediate a wide range of environmental contaminants. Information about which community members respond to stimulation can guide the interpretation and development of remediation approaches. To comprehensively determine community membership and abundance patterns among a suite of samples associated with uranium bioremediation experiments we employed a high-density microarray (PhyloChip). Samples were unstimulated, naturally reducing, or collected during Fe(III) (early) and sulfate reduction (late biostimulation) from an acetate re-amended/amended aquifer in Rifle, Colorado, and from laboratory experiments using field-collected materials. Deep community sampling with PhyloChip identified hundreds-to-thousands of operational taxonomic units (OTUs) present during amendment, and revealed close similarity among highly enriched taxa from drill-core and groundwater well-deployed column sediment. Overall, phylogenetic data suggested stimulated community membership was most affected by a carryover effect between annual stimulation events. Nevertheless, OTUs within the Fe(III)- and sulfate-reducing lineages, Desulfuromonadales and Desulfobacterales, were repeatedly stimulated. Less consistent, co-enriched taxa represented additional lineages associated with Fe(III) and sulfate reduction (for example, Desulfovibrionales; Syntrophobacterales; Peptococcaceae) and autotrophic sulfur oxidation (Sulfurovum; Campylobacterales). These data imply complex membership among highly stimulated taxa, and by inference biogeochemical responses to acetate, a non-fermentable substrate.

  6. Unexpected Advice for Beginning Graduate Students in Astrophysics

    NASA Astrophysics Data System (ADS)

    Linsky, Jeffrey L.

    2012-08-01

    My experience is that beginning graduate students in astrophysics have unrealistic views of how to negotiate the complexities of graduate school and to prepare themselves for a professional career in astrophysics or some other field. This chapter describes my unexpected advice to students beginning with why they should not plan to write a thesis. Other advice concerns how to find and work with a research supervisor, writing and other skills needed for their research, and the need to be creative and when necessary controversial.

  7. Fine mapping of type 1 diabetes regions Idd9.1 and Idd9.2 reveals genetic complexity.

    PubMed

    Hamilton-Williams, Emma E; Rainbow, Daniel B; Cheung, Jocelyn; Christensen, Mikkel; Lyons, Paul A; Peterson, Laurence B; Steward, Charles A; Sherman, Linda A; Wicker, Linda S

    2013-10-01

    Nonobese diabetic (NOD) mice congenic for C57BL/10 (B10)-derived genes in the Idd9 region of chromosome 4 are highly protected from type 1 diabetes (T1D). Idd9 has been divided into three protective subregions (Idd9.1, 9.2, and 9.3), each of which partially prevents disease. In this study we have fine-mapped the Idd9.1 and Idd9.2 regions, revealing further genetic complexity with at least two additional subregions contributing to protection from T1D. Using the NOD sequence from bacterial artificial chromosome clones of the Idd9.1 and Idd9.2 regions as well as whole-genome sequence data recently made available, sequence polymorphisms within the regions highlight a high degree of polymorphism between the NOD and B10 strains in the Idd9 regions. Among numerous candidate genes are several with immunological importance. The Idd9.1 region has been separated into Idd9.1 and Idd9.4, with Lck remaining a candidate gene within Idd9.1. One of the Idd9.2 regions contains the candidate genes Masp2 (encoding mannan-binding lectin serine peptidase 2) and Mtor (encoding mammalian target of rapamycin). From mRNA expression analyses, we have also identified several other differentially expressed candidate genes within the Idd9.1 and Idd9.2 regions. These findings highlight that multiple, relatively small genetic effects combine and interact to produce significant changes in immune tolerance and diabetes onset. PMID:23934554

  8. Complex patterns of mating revealed in a Eucalyptus regnans seed orchard using allozyme markers and the neighbourhood model.

    PubMed

    Burczyk, J; Adams, W T; Moran, G F; Griffin, A R

    2002-11-01

    The neighbourhood model apportions offspring of individual mother plants to self-fertilization, outcrossing to males within a circumscribed area around the mother plant (the neighbourhood), and outcrossing to males outside the neighbourhood. Formerly the model was applied only to haploid pollen gametes in the offspring of conifers, but is extended so that it can be used with genotypic data from diploid offspring of both angiosperms and gymnosperms. In addition, it is shown that the mating parameters can be estimated without independent estimates of allele frequencies in the pollen pools outside the neighbourhood; thus the model might be applied effectively to natural populations exposed to unknown external pollen sources. Parameters of the neighbourhood mating model were estimated for a 10-year-old seed orchard population of the insect-pollinated tree, Eucalyptus regnans, in southeast Australia, which contained a mixture of two geographical provenances (Victoria and Tasmania). The mating patterns revealed were complex. Crosses between trees of the same provenance occurred three times more often than crosses between trees of different provenances. Levels of self-fertilization and patterns of mating within neighbourhoods were influenced by provenance origin, crop fecundity and orchard position (central vs. edge) of mother trees. Gene dispersal, however, was extensive, with approximately 50% of effective pollen gametes coming from males more than 40 m away from mother trees (average distance between neighbouring trees was 7.4 m). Thus, insect pollinators are efficient promoters of cross-fertilization in this orchard, with the result that the effective number of males mating with each female is large. PMID:12406248

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

    USGS Publications Warehouse

    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.

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

    PubMed Central

    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

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

    PubMed

    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

  12. Circadian variation in unexpected postoperative death.

    PubMed

    Rosenberg, J; Pedersen, M H; Ramsing, T; Kehlet, H

    1992-12-01

    Unexpected deaths still occur following major surgical procedures. The cause is often unknown but may be cardiac or thromboembolic in nature. Postoperative ischaemia, infarction and sudden cardiac death may be triggered by episodic or constant arterial hypoxaemia, which increases during the night. This study examined the circadian variation of sudden unexpected death following abdominal surgery between 1985 and 1989 inclusive. Deaths were divided into those occurring during the day (08.00-16.00 hours), evening (16.00-24.00 hours) and night (24.00-08.00 hours). Twenty-three deaths were considered to have been totally unexpected. Of 16 such patients undergoing autopsy, pulmonary embolism was the cause of death in five. In the remaining 11 patients, death occurred at night in eight (P < 0.005). Five of the seven patients without an autopsy died at night (P < 0.04); overall, 13 of 18 unexpected deaths occurred at night-time. These results suggest a need for further studies of sleep- and respiration-related effects on postoperative nocturnal cardiac function. The efficacy of monitoring during this apparent high-risk period should be evaluated. PMID:1486424

  13. Some Unexpected Results Using Computer Algebra Systems.

    ERIC Educational Resources Information Center

    Alonso, Felix; Garcia, Alfonsa; Garcia, Francisco; Hoya, Sara; Rodriguez, Gerardo; de la Villa, Agustin

    2001-01-01

    Shows how teachers can often use unexpected outputs from Computer Algebra Systems (CAS) to reinforce concepts and to show students the importance of thinking about how they use the software and reflecting on their results. Presents different examples where DERIVE, MAPLE, or Mathematica does not work as expected and suggests how to use them as a…

  14. An Unexpected Influence on a Quadratic

    ERIC Educational Resources Information Center

    Davis, Jon D.

    2013-01-01

    Using technology to explore the coefficients of a quadratic equation can lead to an unexpected result. This article describes an investigation that involves sliders and dynamically linked representations. It guides students to notice the effect that the parameter "a" has on the graphical representation of a quadratic function in the form…

  15. Exploiting Unexpected Situations in the Mathematics Classroom

    ERIC Educational Resources Information Center

    Foster, Colin

    2015-01-01

    The professional development of mathematics teachers needs to support teachers in orchestrating the mathematics classroom in ways that enable them to respond flexibly and productively to the unexpected. When a situation arises in the classroom which is not connected in an obvious way to the mathematical learning intentions of the lesson, it can be…

  16. Functional dissection of the NuA4 histone acetyltransferase reveals its role as a genetic hub and that Eaf1 is essential for complex integrity.

    PubMed

    Mitchell, Leslie; Lambert, Jean-Philippe; Gerdes, Maria; Al-Madhoun, Ashraf S; Skerjanc, Ilona S; Figeys, Daniel; Baetz, Kristin

    2008-04-01

    The Saccharomyces cerevisiae NuA4 histone acetyltransferase complex catalyzes the acetylation of histone H4 and the histone variant Htz1 to regulate key cellular events, including transcription, DNA repair, and faithful chromosome segregation. To further investigate the cellular processes impacted by NuA4, we exploited the nonessential subunits of the complex to build an extensive NuA4 genetic-interaction network map. The map reveals that NuA4 is a genetic hub whose function buffers a diverse range of cellular processes, many not previously linked to the complex, including Golgi complex-to-vacuole vesicle-mediated transport. Further, we probe the role that nonessential subunits play in NuA4 complex integrity. We find that most nonessential subunits have little impact on NuA4 complex integrity and display between 12 and 42 genetic interactions. In contrast, the deletion of EAF1 causes the collapse of the NuA4 complex and displays 148 genetic interactions. Our study indicates that Eaf1 plays a crucial function in NuA4 complex integrity. Further, we determine that Eaf5 and Eaf7 form a subcomplex, which reflects their similar genetic interaction profiles and phenotypes. Our integrative study demonstrates that genetic interaction maps are valuable in dissecting complex structure and provides insight into why the human NuA4 complex, Tip60, has been associated with a diverse range of pathologies. PMID:18212056

  17. Electrophoretic behavior of DNA-methyl-CpG-binding domain protein complexes revealed by capillary electrophoreses laser-induced fluorescence.

    PubMed

    Zhong, Shangwei; Zou, Dandan; Zhao, Bailin; Zhang, Dapeng; Li, Xiangjun; Wang, Hailin

    2015-12-01

    The free solution electrophoretic behavior of DNA-protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA-protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA-methyl-CpG-binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA-protein complexes. This method is based on the effective separation of free DNA and DNA-protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide-coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA-MBD2b complexes, we found that the values of (μ0 /μ)-1 were directly proportional to the charge-to-mass ratios of formed complexes, where the μ0 and μ are the mobility of free DNA probe and DNA-protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge-to-mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA-binding properties of the purified MBD2b protein and help to analyze other DNA-protein complexes. Additionally, this study enhances the understanding of the influence of the charge-to-mass ratios of formed DNA-protein complexes on their separation and electrophoretic behaviors. PMID:26377303

  18. Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation

    PubMed Central

    Zhang, Yi; Ng, Huck-Hui; Erdjument-Bromage, Hediye; Tempst, Paul; Bird, Adrian; Reinberg, Danny

    1999-01-01

    ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation. PMID:10444591

  19. Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex

    SciTech Connect

    Žáková, Lenka; Kletvíková, Emília; Lepšík, Martin; Collinsová, Michaela; Watson, Christopher J.; Turkenburg, Johan P.; Jiráček, Jiří; Brzozowski, Andrzej M.

    2014-10-01

    [AsnB26]- and [GlyB26]-insulin mutants attain a B26-turn like fold without assistance of chemical modifications. Their structures match the insulin receptor interface and expand the spectrum of insulin conformations. The structural characterization of the insulin–insulin receptor (IR) interaction still lacks the conformation of the crucial B21–B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that in all known structures of the native hormone. It also matches the receptor interface, avoiding substantial steric clashes. This indicates that insulin may attain a B26-turn-like conformation upon IR binding. Moreover, there is an unexpected, but significant, binding specificity of the AsnB26 mutant for predominantly the metabolic B isoform of the receptor. As it is correlated with the B26 bend of the B-chain of the hormone, the structures of AsnB26 analogues may provide the first structural insight into the structural origins of differential insulin signalling through insulin receptor A and B isoforms.

  20. Distinct roles of apolipoprotein components within the trypanosome lytic factor complex revealed in a novel transgenic mouse model.

    PubMed

    Molina-Portela, Maria Pilar; Samanovic, Marie; Raper, Jayne

    2008-08-01

    Humans express a unique subset of high-density lipoproteins (HDLs) called trypanosome lytic factors (TLFs) that kill many Trypanosoma parasite species. The proteins apolipoprotein (apo) A-I, apoL-I, and haptoglobin-related protein, which are involved in TLF structure and function, were expressed through the introduction of transgenes in mice to explore their physiological roles in vivo. Transgenic expression of human apolipoprotein L-I alone conferred trypanolytic activity in vivo. Coexpression of human apolipoprotein A-I and haptoglobin-related protein (Hpr) had an effect on the integration of apolipoprotein L-I into HDL, and both proteins were required to increase the specific activity of TLF, which was measurable in vitro. Unexpectedly, truncated apolipoprotein L-I devoid of the serum resistance gene interacting domain, which was previously shown to kill human infective trypanosomes, was not trypanolytic in transgenic mice despite being coexpressed with human apolipoprotein A-I and Hpr and incorporated into HDLs. We conclude that all three human apolipoproteins act cooperatively to achieve maximal killing capacity and that truncated apolipoprotein L-I does not function in transgenic animals. PMID:18606856

  1. Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex

    PubMed Central

    Žáková, Lenka; Kletvíková, Emília; Lepšík, Martin; Collinsová, Michaela; Watson, Christopher J.; Turkenburg, Johan P.; Jiráček, Jiří; Brzozowski, Andrzej M.

    2014-01-01

    The structural characterization of the insulin–insulin receptor (IR) interaction still lacks the conformation of the crucial B21–B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that in all known structures of the native hormone. It also matches the receptor interface, avoiding substantial steric clashes. This indicates that insulin may attain a B26-turn-like conformation upon IR binding. Moreover, there is an unexpected, but significant, binding specificity of the AsnB26 mutant for predominantly the metabolic B isoform of the receptor. As it is correlated with the B26 bend of the B-chain of the hormone, the structures of AsnB26 analogues may provide the first structural insight into the structural origins of differential insulin signalling through insulin receptor A and B isoforms. PMID:25286859

  2. Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex.

    PubMed

    Záková, Lenka; Kletvíková, Emília; Lepšík, Martin; Collinsová, Michaela; Watson, Christopher J; Turkenburg, Johan P; Jiráček, Jiří; Brzozowski, Andrzej M

    2014-10-01

    The structural characterization of the insulin-insulin receptor (IR) interaction still lacks the conformation of the crucial B21-B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that in all known structures of the native hormone. It also matches the receptor interface, avoiding substantial steric clashes. This indicates that insulin may attain a B26-turn-like conformation upon IR binding. Moreover, there is an unexpected, but significant, binding specificity of the AsnB26 mutant for predominantly the metabolic B isoform of the receptor. As it is correlated with the B26 bend of the B-chain of the hormone, the structures of AsnB26 analogues may provide the first structural insight into the structural origins of differential insulin signalling through insulin receptor A and B isoforms. PMID:25286859

  3. Structural and functional characterization of a cell cycle associated HDAC1/2 complex reveals the structural basis for complex assembly and nucleosome targeting

    PubMed Central

    Itoh, Toshimasa; Fairall, Louise; Muskett, Frederick W.; Milano, Charles P.; Watson, Peter J.; Arnaudo, Nadia; Saleh, Almutasem; Millard, Christopher J.; El-Mezgueldi, Mohammed; Martino, Fabrizio; Schwabe, John W.R.

    2015-01-01

    Recent proteomic studies have identified a novel histone deacetylase complex that is upregulated during mitosis and is associated with cyclin A. This complex is conserved from nematodes to man and contains histone deacetylases 1 and 2, the MIDEAS corepressor protein and a protein called DNTTIP1 whose function was hitherto poorly understood. Here, we report the structures of two domains from DNTTIP1. The amino-terminal region forms a tight dimerization domain with a novel structural fold that interacts with and mediates assembly of the HDAC1:MIDEAS complex. The carboxy-terminal domain of DNTTIP1 has a structure related to the SKI/SNO/DAC domain, despite lacking obvious sequence homology. We show that this domain in DNTTIP1 mediates interaction with both DNA and nucleosomes. Thus, DNTTIP1 acts as a dimeric chromatin binding module in the HDAC1:MIDEAS corepressor complex. PMID:25653165

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

    PubMed Central

    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

  5. Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex*

    PubMed Central

    Shi, Yi; Fernandez-Martinez, Javier; Tjioe, Elina; Pellarin, Riccardo; Kim, Seung Joong; Williams, Rosemary; Schneidman-Duhovny, Dina; Sali, Andrej; Rout, Michael P.; Chait, Brian T.

    2014-01-01

    Most cellular processes are orchestrated by macromolecular complexes. However, structural elucidation of these endogenous complexes can be challenging because they frequently contain large numbers of proteins, are compositionally and morphologically heterogeneous, can be dynamic, and are often of low abundance in the cell. Here, we present a strategy for the structural characterization of such complexes that has at its center chemical cross-linking with mass spectrometric readout. In this strategy, we isolate the endogenous complexes using a highly optimized sample preparation protocol and generate a comprehensive, high-quality cross-linking dataset using two complementary cross-linking reagents. We then determine the structure of the complex using a refined integrative method that combines the cross-linking data with information generated from other sources, including electron microscopy, X-ray crystallography, and comparative protein structure modeling. We applied this integrative strategy to determine the structure of the native Nup84 complex, a stable hetero-heptameric assembly (∼600 kDa), 16 copies of which form the outer rings of the 50-MDa nuclear pore complex (NPC) in budding yeast. The unprecedented detail of the Nup84 complex structure reveals previously unseen features in its pentameric structural hub and provides information on the conformational flexibility of the assembly. These additional details further support and augment the protocoatomer hypothesis, which proposes an evolutionary relationship between vesicle coating complexes and the NPC, and indicates a conserved mechanism by which the NPC is anchored in the nuclear envelope. PMID:25161197

  6. Inducible Repression of Nuclear-Encoded Subunits of the Cytochrome b6f Complex in Tobacco Reveals an Extraordinarily Long Lifetime of the Complex1[W][OPEN

    PubMed Central

    Hojka, Marta; Thiele, Wolfram; Tóth, Szilvia Z.; Lein, Wolfgang; Bock, Ralph; Schöttler, Mark Aurel

    2014-01-01

    The biogenesis of the cytochrome b6f complex in tobacco (Nicotiana tabacum) seems to be restricted to young leaves, suggesting a high lifetime of the complex. To directly determine its lifetime, we employed an ethanol-inducible RNA interference (RNAi) approach targeted against the essential nuclear-encoded Rieske protein (PetC) and the small M subunit (PetM), whose function in higher plants is unknown. Young expanding leaves of both PetM and PetC RNAi transformants bleached rapidly and developed necroses, while mature leaves, whose photosynthetic apparatus was fully assembled before RNAi induction, stayed green. In line with these phenotypes, cytochrome b6f complex accumulation and linear electron transport capacity were strongly repressed in young leaves of both RNAi transformants, showing that the M subunit is as essential for cytochrome b6f complex accumulation as the Rieske protein. In mature leaves, all photosynthetic parameters were indistinguishable from the wild type even after 14 d of induction. As RNAi repression of PetM and PetC was highly efficient in both young and mature leaves, these data indicate a lifetime of the cytochrome b6f complex of at least 1 week. The switch-off of cytochrome b6f complex biogenesis in mature leaves may represent part of the first dedicated step of the leaf senescence program. PMID:24963068

  7. Crystal structure of the Chlamydomonas starch debranching enzyme isoamylase ISA1 reveals insights into the mechanism of branch trimming and complex assembly.

    PubMed

    Sim, Lyann; Beeren, Sophie R; Findinier, Justin; Dauvillée, David; Ball, Steven G; Henriksen, Anette; Palcic, Monica M

    2014-08-15

    The starch debranching enzymes isoamylase 1 and 2 (ISA1 and ISA2) are known to exist in a large complex and are involved in the biosynthesis and crystallization of starch. It is suggested that the function of the complex is to remove misplaced branches of growing amylopectin molecules, which would otherwise prevent the association and crystallization of adjacent linear chains. Here, we investigate the function of ISA1 and ISA2 from starch producing alga Chlamydomonas. Through complementation studies, we confirm that the STA8 locus encodes for ISA2 and sta8 mutants lack the ISA1·ISA2 heteromeric complex. However, mutants retain a functional dimeric ISA1 that is able to partly sustain starch synthesis in vivo. To better characterize ISA1, we have overexpressed and purified ISA1 from Chlamydomonas reinhardtii (CrISA1) and solved the crystal structure to 2.3 Å and in complex with maltoheptaose to 2.4 Å. Analysis of the homodimeric CrISA1 structure reveals a unique elongated structure with monomers connected end-to-end. The crystal complex reveals details about the mechanism of branch binding that explains the low activity of CrISA1 toward tightly spaced branches and reveals the presence of additional secondary surface carbohydrate binding sites. PMID:24993830

  8. A Developmental Framework for Complex Plasmodesmata Formation Revealed by Large-Scale Imaging of the Arabidopsis Leaf Epidermis[W

    PubMed Central

    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. PMID:23371949

  9. Characterization of the Mycobacterial Acyl-CoA Carboxylase Holo Complexes Reveals Their Functional Expansion into Amino Acid Catabolism

    PubMed Central

    Ehebauer, Matthias T.; Zimmermann, Michael; Jakobi, Arjen J.; Noens, Elke E.; Laubitz, Daniel; Cichocki, Bogdan; Marrakchi, Hedia; Lanéelle, Marie-Antoinette; Daffé, Mamadou; Sachse, Carsten; Dziembowski, Andrzej; Sauer, Uwe; Wilmanns, Matthias

    2015-01-01

    Biotin-mediated carboxylation of short-chain fatty acid coenzyme A esters is a key step in lipid biosynthesis that is carried out by multienzyme complexes to extend fatty acids by one methylene group. Pathogenic mycobacteria have an unusually high redundancy of carboxyltransferase genes and biotin carboxylase genes, creating multiple combinations of protein/protein complexes of unknown overall composition and functional readout. By combining pull-down assays with mass spectrometry, we identified nine binary protein/protein interactions and four validated holo acyl-coenzyme A carboxylase complexes. We investigated one of these - the AccD1-AccA1 complex from Mycobacterium tuberculosis with hitherto unknown physiological function. Using genetics, metabolomics and biochemistry we found that this complex is involved in branched amino-acid catabolism with methylcrotonyl coenzyme A as the substrate. We then determined its overall architecture by electron microscopy and found it to be a four-layered dodecameric arrangement that matches the overall dimensions of a distantly related methylcrotonyl coenzyme A holo complex. Our data argue in favor of distinct structural requirements for biotin-mediated γ-carboxylation of α−β unsaturated acid esters and will advance the categorization of acyl-coenzyme A carboxylase complexes. Knowledge about the underlying structural/functional relationships will be crucial to make the target category amenable for future biomedical applications. PMID:25695631

  10. Sudden Unexpected Postnatal Collapse of the Newborn.

    PubMed

    Ferrarello, Debi; Carmichael, Tanya

    2016-01-01

    Sudden unexpected postnatal collapse is a rare but devastating neonatal event. A well-appearing, full-term newborn with Agpar scores of eight or more suddenly crashes, often with full respiratory and cardiac arrest. Up to half of newborns with sudden unexpected postnatal collapse die, with many survivors suffering serious neurological damage. The first 2 hours of life are the hours of greatest risk, coinciding with the time frame when nurses encourage breastfeeding and uninterrupted skin-to-skin contact between women and newborns. Nursing assessments and measures to promote neonates' optimal transition to extrauterine life through skin-to-skin contact and early breastfeeding while decreasing the risk of this catastrophic event are described. Nursing surveillance to promote optimal transition in a safe environment is essential, and birth facilities should allocate staffing resources accordingly. PMID:27287353

  11. BaBar: Searching for the Unexpected

    SciTech Connect

    Roodman, Aaron

    2006-04-24

    With over 300 million B-Bbar pairs delivered by the PEP-II B-factory, and over 200 publications, the Babar experiment has studied a very broad range of B, charm, tau, and QCD topics. In fact, the physics at Babar is so wide-ranging that it threatens to overwhelm any summary. Instead, in this colloquium I will focus on just a few topics selected for their potential to find a truly unexpected result. These include CP violation in rare B decays, leptonic B decays, and lepton-flavor violating tau decays. Each measurement, the currently predicted result, and the potential for uncovering the unexpected now and into the future, will be gently, but thoroughly, described.

  12. Regulatory RNAs discovered in unexpected places.

    PubMed

    Pek, Jun Wei; Okamura, Katsutomo

    2015-01-01

    Recent studies have discovered both small and long noncoding RNAs (ncRNAs) encoded in unexpected places. These ncRNA genes were surprises at the time of their discovery, but many quickly became well-accepted families of functional regulatory RNA species. Even after years of extensive gene annotation studies using high-throughput sequencing technologies, new types of ncRNA genes continue to be discovered in unexpected places. We highlight ncRNAs that have atypical structures and that are encoded in what are generally considered 'junk' sequences, such as spacers and introns. We also discuss current bottlenecks in the approaches for identifying novel ncRNAs and the possibility that many remain to be discovered. PMID:26424536

  13. [Neurosyphilis: unexpected reunion with an old acquaintance].

    PubMed

    Lens-Daey Ouwens, I; Heijstra, M P; Timmerman, L

    2011-01-01

    A 45-year-old man was admitted to a psychiatric hospital with confusion and disorientation; he was suspected of having Korsakoff syndrome. He was known to have a history of alcohol abuse, complicated by epileptic fits, and to have had a recent ischaemic cerebrovascular attack. Unexpectedly, screening for syphilis turned out to be positive. Examination of the cerebrospinal fluid led to the diagnosis of neurosyphilis. Most neurological and psychiatric symptoms disappeared after treatment with antibiotics. PMID:21319069

  14. Illustrations of Unexpected Infant Sleep Deaths.

    PubMed

    Cullen, Deborah; Oberle, Morgan; Elomba, Charles D; Stiffler, Deborah; Luna, Gaye

    2016-01-01

    Case illustrations from central Indiana provide the narrative for infant suffocations because of unsafe sleep environments. Accidental strangulation or suffocation in bed is caused by co-bedding, blankets and pillows in cribs, or wedging and entrapment. Knowledge of the evidence-based risks associated with case data may assist further in the prevention of unexpected infant sleep deaths and may better inform best practice for death scene investigation including forensic nurses. PMID:27496648

  15. Revealing rate-limiting steps in complex disease biology: The crucial importance of studying rare, extreme-phenotype families.

    PubMed

    Chakravarti, Aravinda; Turner, Tychele N

    2016-06-01

    The major challenge in complex disease genetics is to understand the fundamental features of this complexity and why functional alterations at multiple independent genes conspire to lead to an abnormal phenotype. We hypothesize that the various genes involved are all functionally united through gene regulatory networks (GRN), and that mutant phenotypes arise from the consequent perturbation of one or more rate-limiting steps that affect the function of the entire GRN. Understanding a complex phenotype thus entails unraveling the details of each GRN, namely, the transcription factors that bind to cis regulatory elements affected by sequence variants altering transcription of specific genes, and their mutual feedback relationships. These GRNs can be identified through their rate-limiting steps and are best uncovered by genomic analyses of rare, extreme phenotype families, thus providing a coherent molecular basis to complex traits and disorders. PMID:27062178

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

    PubMed

    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. PMID:26381410

  17. Unexpected involvement of staple leads to redesign of selective bicyclic peptide inhibitor of Grb7

    NASA Astrophysics Data System (ADS)

    Gunzburg, Menachem J.; Kulkarni, Ketav; Watson, Gabrielle M.; Ambaye, Nigus D.; Del Borgo, Mark P.; Brandt, Rebecca; Pero, Stephanie C.; Perlmutter, Patrick; Wilce, Matthew C. J.; Wilce, Jacqueline A.

    2016-06-01

    The design of potent and specific peptide inhibitors to therapeutic targets is of enormous utility for both proof-of-concept studies and for the development of potential new therapeutics. Grb7 is a key signaling molecule in the progression of HER2 positive and triple negative breast cancers. Here we report the crystal structure of a stapled bicyclic peptide inhibitor G7-B1 in complex with the Grb7-SH2 domain. This revealed an unexpected binding mode of the peptide, in which the staple forms an alternative contact with the surface of the target protein. Based on this structural information, we designed a new series of bicyclic G7 peptides that progressively constrain the starting peptide, to arrive at the G7-B4 peptide that binds with an approximately 2-fold enhanced affinity to the Grb7-SH2 domain (KD = 0.83 μM) compared to G7-B1 and shows low affinity binding to Grb2-, Grb10- and Grb14-SH2 domains (KD > 100 μM). Furthermore, we determined the structure of the G7-B4 bicyclic peptide in complex with the Grb7-SH2 domain, both before and after ring closing metathesis to show that the closed staple is essential to the target interaction. The G7-B4 peptide represents an advance in the development of Grb7 inhibitors and is a classical example of structure aided inhibitor development.

  18. Unexpected involvement of staple leads to redesign of selective bicyclic peptide inhibitor of Grb7.

    PubMed

    Gunzburg, Menachem J; Kulkarni, Ketav; Watson, Gabrielle M; Ambaye, Nigus D; Del Borgo, Mark P; Brandt, Rebecca; Pero, Stephanie C; Perlmutter, Patrick; Wilce, Matthew C J; Wilce, Jacqueline A

    2016-01-01

    The design of potent and specific peptide inhibitors to therapeutic targets is of enormous utility for both proof-of-concept studies and for the development of potential new therapeutics. Grb7 is a key signaling molecule in the progression of HER2 positive and triple negative breast cancers. Here we report the crystal structure of a stapled bicyclic peptide inhibitor G7-B1 in complex with the Grb7-SH2 domain. This revealed an unexpected binding mode of the peptide, in which the staple forms an alternative contact with the surface of the target protein. Based on this structural information, we designed a new series of bicyclic G7 peptides that progressively constrain the starting peptide, to arrive at the G7-B4 peptide that binds with an approximately 2-fold enhanced affinity to the Grb7-SH2 domain (KD = 0.83 μM) compared to G7-B1 and shows low affinity binding to Grb2-, Grb10- and Grb14-SH2 domains (KD > 100 μM). Furthermore, we determined the structure of the G7-B4 bicyclic peptide in complex with the Grb7-SH2 domain, both before and after ring closing metathesis to show that the closed staple is essential to the target interaction. The G7-B4 peptide represents an advance in the development of Grb7 inhibitors and is a classical example of structure aided inhibitor development. PMID:27257138

  19. Unexpected involvement of staple leads to redesign of selective bicyclic peptide inhibitor of Grb7

    PubMed Central

    Gunzburg, Menachem J.; Kulkarni, Ketav; Watson, Gabrielle M.; Ambaye, Nigus D.; Del Borgo, Mark P.; Brandt, Rebecca; Pero, Stephanie C.; Perlmutter, Patrick; Wilce, Matthew C. J.; Wilce, Jacqueline A.

    2016-01-01

    The design of potent and specific peptide inhibitors to therapeutic targets is of enormous utility for both proof-of-concept studies and for the development of potential new therapeutics. Grb7 is a key signaling molecule in the progression of HER2 positive and triple negative breast cancers. Here we report the crystal structure of a stapled bicyclic peptide inhibitor G7-B1 in complex with the Grb7-SH2 domain. This revealed an unexpected binding mode of the peptide, in which the staple forms an alternative contact with the surface of the target protein. Based on this structural information, we designed a new series of bicyclic G7 peptides that progressively constrain the starting peptide, to arrive at the G7-B4 peptide that binds with an approximately 2-fold enhanced affinity to the Grb7-SH2 domain (KD = 0.83 μM) compared to G7-B1 and shows low affinity binding to Grb2-, Grb10- and Grb14-SH2 domains (KD > 100 μM). Furthermore, we determined the structure of the G7-B4 bicyclic peptide in complex with the Grb7-SH2 domain, both before and after ring closing metathesis to show that the closed staple is essential to the target interaction. The G7-B4 peptide represents an advance in the development of Grb7 inhibitors and is a classical example of structure aided inhibitor development. PMID:27257138

  20. In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes[OPEN

    PubMed Central

    Husbands, Aman Y.; Aggarwal, Vasudha; Ha, Taekjip; Timmermans, Marja C.P.

    2016-01-01

    Deciphering complex biological processes markedly benefits from approaches that directly assess the underlying biomolecular interactions. Most commonly used approaches to monitor protein-protein interactions typically provide nonquantitative readouts that lack statistical power and do not yield information on the heterogeneity or stoichiometry of protein complexes. Single-molecule pull-down (SiMPull) uses single-molecule fluorescence detection to mitigate these disadvantages and can quantitatively interrogate interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids. Here, we establish SiMPull in plants using the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) and LITTLE ZIPPER (ZPR) interaction as proof-of-principle. Colocalization analysis of fluorophore-tagged HD-ZIPIII and ZPR proteins provides strong statistical evidence of complex formation. In addition, we use SiMPull to directly quantify YFP and mCherry maturation probabilities, showing these differ substantially from values obtained in mammalian systems. Leveraging these probabilities, in conjunction with fluorophore photobleaching assays on over 2000 individual complexes, we determined HD-ZIPIII:ZPR stoichiometry. Intriguingly, these complexes appear as heterotetramers, comprising two HD-ZIPIII and two ZPR molecules, rather than heterodimers as described in the current model. This surprising result raises new questions about the regulation of these key developmental factors and is illustrative of the unique contribution SiMPull is poised to make to in planta protein interaction studies. PMID:27385814

  1. Unexpected reactivity of 2-fluorolinalyl diphosphate in the active site of crystalline 2-methylisoborneol synthase

    PubMed Central

    Köksal, Mustafa; Chou, Wayne K. W.; Cane, David E.; Christianson, David W.

    2013-01-01

    The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in its unliganded state and in complex with 2 Mg2+ ions and cis-2-fluorogeranyl diphosphate at 1.85 Å and 2.00 Å resolution, respectively. Under normal circumstances, MIBS catalyzes the cyclization of the naturally-occurring, non-canonical 11-carbon isoprenoid substrate, 2-methylgeranyl diphosphate, which first undergoes an ionization-isomerization-ionization sequence through the tertiary diphosphate intermediate 2-methyllinalyl diphosphate to enable subsequent cyclization chemistry. MIBS does not exhibit catalytic activity with 2-fluorogeranyl diphosphate, and we recently reported the crystal structure of MIBS complexed with this unreactive substrate analogue [Köksal, M., Chou, W. K. W., Cane, D. E., Christianson, D. W. (2012) Biochemistry 51, 3011–3020]. However, cocrystallization of MIBS with the fluorinated analogue of the tertiary allylic diphosphate intermediate, 2-fluorolinalyl diphosphate, reveals unexpected reactivity for the intermediate analogue and yields the crystal structure of the complex with the primary allylic diphosphate, 2-fluoroneryl diphosphate. Comparison with the structure of the unliganded enzyme reveals that the crystalline enzyme active site remains partially open, presumably due to the binding of only 2 Mg2+ ions. Assays in solution indicate that MIBS catalyzes the generation of (1R)-(+)-camphor from the substrate 2-fluorolinalyl diphosphate, suggesting that both 2-fluorolinalyl diphosphate and 2-methyllinalyl diphosphate follow the identical cyclization mechanism leading to 2-substituted isoborneol products; however, the initially generated 2-fluoroisoborneol cyclization product is unstable and undergoes elimination of hydrogen fluoride to yield (1R)-(+)-camphor. PMID:23844678

  2. An unexpected twist in viral capsid maturation

    SciTech Connect

    Gertsman, Ilya; Gan, Lu; Guttman, Miklos; Lee, Kelly; Speir, Jeffrey A.; Duda, Robert L.; Hendrix, Roger W.; Komives, Elizabeth A.; Johnson, John E.

    2009-04-14

    Lambda-like double-stranded (ds) DNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging. The extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Although various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage. Here we present a procapsid X-ray structure at 3.65 {angstrom} resolution, termed prohead II, of the lambda-like bacteriophage HK97, the mature capsid structure of which was previously solved to 3.44 {angstrom}. A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and hydrogen/deuterium exchange data presented here demonstrate that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and -sheet regions. We also identified subunit interactions at each three-fold axis of the capsid that are maintained throughout maturation. The interactions sustain capsid integrity during subunit refolding and provide a fixed hinge from which subunits undergo rotational and translational motions during maturation. Previously published calorimetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90 kJ mol{sup -1} of energy. We propose that the major tertiary changes presented in this study reveal a structural basis for an exothermic

  3. An Unexpected Twist in Viral Capsid Maturation

    PubMed Central

    Gertsman, Ilya; Gan, Lu; Guttman, Miklos; Lee, Kelly; Speir, Jeffrey A.; Duda, Robert L.; Hendrix, Roger W.; Komives, Elizabeth A.; Johnson, John E.

    2009-01-01

    Lambda-like dsDNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm. of pressure during genome packaging1. The extensive integration between subunits in capsids is unlikely to form in a single assembly step, therefore requiring formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Though various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage that undergoes large scale conformational changes. We present a procapsid x-ray structure at 3.65Å resolution, termed Prohead II, of the lambda like bacteriophage HK97, whose mature capsid structure was previously solved to 3.44 Å2. A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and Hydrogen/Deuterium exchange data presented here demonstrates that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and β-sheet regions. We have also discovered conserved subunit interactions at each 3-fold of the virus capsid, from which capsid subunits maintain their integrity during refolding, facilitating the rotational and translational motions of maturation. Calormetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90KJ/mol of energy3. We propose the major tertiary changes presented in this study reveal a structural basis for an exothermic maturation process likely present in many ds

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

    PubMed Central

    2012-01-01

    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 understanding how parasite

  5. Stretched peer-review on unexpected results (GMOs).

    PubMed

    Myhr, A I

    2005-01-01

    Science is the basis for governance of risk from genetically modified organisms (GMO), and it is also a primary source of legitimacy for policy decision. However, recently the publication of unexpected results has caused controversies and challenged the way in which science should be performed, be published in scientific journals, and how preliminary results should be communicated. These studies have subsequently, after being accepted for publication within the peer-review process of leading scientific journals, been thoroughly re-examined by many actors active within the GMO debate and thereby drawn extensive media coverage. The publicized charges that the research involved does not constitute significant evidence or represent bad science have in fact deflected attention away from the important questions related to ecological and health risks raised by the research. In this paper, I will argue that unexpected findings may represent "early warnings." Although early warnings may not represent reality, such reports are necessary to inform other scientists and regulators, and should be followed up by further research to reveal the validity of the warnings. Furthermore, science that embraces robust, participatory and transparent approaches will be imperative in the future to reduce the present controversy surrounding GMO use and release. PMID:16304941

  6. Structure of a helicase–helicase loader complex reveals insights into the mechanism of bacterial primosome assembly

    SciTech Connect

    Liu, Bin; Eliason, William K.; Steitz, Thomas A.

    2013-09-19

    During the assembly of the bacterial loader-dependent primosome, helicase loader proteins bind to the hexameric helicase ring, deliver it onto the oriC DNA and then dissociate from the complex. Here, to provide a better understanding of this key process, we report the crystal structure of the ~570-kDa prepriming complex between the Bacillus subtilis loader protein and the Bacillus stearothermophilus helicase, as well as the helicase-binding domain of primase with a molar ratio of 6:6:3 at 7.5 Å resolution. The overall architecture of the complex exhibits a three-layered ring conformation. Moreover, the structure combined with the proposed model suggests that the shift from the ‘open-ring’ to the ‘open-spiral’ and then the ‘closed-spiral’ state of the helicase ring due to the binding of single-stranded DNA may be the cause of the loader release.

  7. In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes*

    PubMed Central

    Clifton, Matthew C.; Simon, Michael J.; Erramilli, Satchal K.; Zhang, Huide; Zaitseva, Jelena; Hermodson, Mark A.; Stauffacher, Cynthia V.

    2015-01-01

    Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg2+. We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg2+, and VO4); a RbsAC complex in the presence of ADP and Mg2+; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB