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Sample records for co-expressing chaperonins groes

  1. Conversion of an inactive xylose isomerase into a functional enzyme by co-expression of GroEL-GroES chaperonins in Saccharomyces cerevisiae.

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    Temer, Beatriz; Dos Santos, Leandro Vieira; Negri, Victor Augusti; Galhardo, Juliana Pimentel; Magalhães, Pedro Henrique Mello; José, Juliana; Marschalk, Cidnei; Corrêa, Thamy Lívia Ribeiro; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante Guimarães

    2017-09-09

    Second-generation ethanol production is a clean bioenergy source with potential to mitigate fossil fuel emissions. The engineering of Saccharomyces cerevisiae for xylose utilization is an essential step towards the production of this biofuel. Though xylose isomerase (XI) is the key enzyme for xylose conversion, almost half of the XI genes are not functional when expressed in S. cerevisiae. To date, protein misfolding is the most plausible hypothesis to explain this phenomenon. This study demonstrated that XI from the bacterium Propionibacterium acidipropionici becomes functional in S. cerevisiae when co-expressed with GroEL-GroES chaperonin complex from Escherichia coli. The developed strain BTY34, harboring the chaperonin complex, is able to efficiently convert xylose to ethanol with a yield of 0.44 g ethanol/g xylose. Furthermore, the BTY34 strain presents a xylose consumption rate similar to those observed for strains carrying the widely used XI from the fungus Orpinomyces sp. In addition, the tetrameric XI structure from P. acidipropionici showed an elevated number of hydrophobic amino acid residues on the surface of protein when compared to XI commonly expressed in S. cerevisiae. Based on our results, we elaborate an extensive discussion concerning the uncertainties that surround heterologous expression of xylose isomerases in S. cerevisiae. Probably, a correct folding promoted by GroEL-GroES could solve some issues regarding a limited or absent XI activity in S. cerevisiae. The strains developed in this work have promising industrial characteristics, and the designed strategy could be an interesting approach to overcome the non-functionality of bacterial protein expression in yeasts.

  2. Myxococcus xanthus DK1622 Coordinates Expressions of the Duplicate groEL and Single groES Genes for Synergistic Functions of GroELs and GroES

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    Yue-zhong Li

    2017-04-01

    Full Text Available Chaperonin GroEL (Cpn60 requires cofactor GroES (Cpn10 for protein refolding in bacteria that possess single groEL and groES genes in a bicistronic groESL operon. Among 4,861 completely-sequenced prokaryotic genomes, 884 possess duplicate groEL genes and 770 possess groEL genes with no neighboring groES. It is unclear whether stand-alone groEL requires groES in order to function and, if required, how duplicate groEL genes and unequal groES genes balance their expressions. In Myxococcus xanthus DK1622, we determined that, while duplicate groELs were alternatively deletable, the single groES that clusters with groEL1 was essential for cell survival. Either GroEL1 or GroEL2 required interactions with GroES for in vitro and in vivo functions. Deletion of groEL1 or groEL2 resulted in decreased expressions of both groEL and groES; and ectopic complementation of groEL recovered not only the groEL but also groES expressions. The addition of an extra groES gene upstream groEL2 to form a bicistronic operon had almost no influence on groES expression and the cell survival rate, whereas over-expression of groES using a self-replicating plasmid simultaneously increased the groEL expressions. The results indicated that M. xanthus DK1622 cells coordinate expressions of the duplicate groEL and single groES genes for synergistic functions of GroELs and GroES. We proposed a potential regulation mechanism for the expression coordination.

  3. In silico engineering of aggregation-prone recombinant proteins for substrate recognition by the chaperonin GroEL

    National Research Council Canada - National Science Library

    Kumar, Vipul; Punetha, Ankita; Sundar, Durai; Chaudhuri, Tapan K

    2012-01-01

    Molecular chaperones appear to have been evolved to facilitate protein folding in the cell through entrapment of folding intermediates on the interior of a large cavity formed between GroEL and its co-chaperonin GroES...

  4. Identification of elements that dictate the specificity of mitochondrial Hsp60 for its co-chaperonin.

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    Avital Parnas

    Full Text Available Type I chaperonins (cpn60/Hsp60 are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES. The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-chaperonin, GroES. We isolated two mutants, a single mutant (E321K and a double mutant (R264K/E358K that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co-chaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian

  5. Difference in the distribution pattern of substrate enzymes in the metabolic network of Escherichia coli, according to chaperonin requirement.

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    Takemoto, Kazuhiro; Niwa, Tatsuya; Taguchi, Hideki

    2011-06-24

    Chaperonins are important in living systems because they play a role in the folding of proteins. Earlier comprehensive analyses identified substrate proteins for which folding requires the chaperonin GroEL/GroES (GroE) in Escherichia coli, and they revealed that many chaperonin substrates are metabolic enzymes. This result implies the importance of chaperonins in metabolism. However, the relationship between chaperonins and metabolism is still unclear. We investigated the distribution of chaperonin substrate enzymes in the metabolic network using network analysis techniques as a first step towards revealing this relationship, and found that as chaperonin requirement increases, substrate enzymes are more laterally distributed in the metabolic. In addition, comparative genome analysis showed that the chaperonin-dependent substrates were less conserved, suggesting that these substrates were acquired later on in evolutionary history. This result implies the expansion of metabolic networks due to this chaperonin, and it supports the existing hypothesis of acceleration of evolution by chaperonins. The distribution of chaperonin substrate enzymes in the metabolic network is inexplicable because it does not seem to be associated with individual protein features such as protein abundance, which has been observed characteristically in chaperonin substrates in previous works. However, it becomes clear by considering this expansion process due to chaperonin. This finding provides new insights into metabolic evolution and the roles of chaperonins in living systems.

  6. Difference in the distribution pattern of substrate enzymes in the metabolic network of Escherichia coli, according to chaperonin requirement

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    Niwa Tatsuya

    2011-06-01

    Full Text Available Abstract Background Chaperonins are important in living systems because they play a role in the folding of proteins. Earlier comprehensive analyses identified substrate proteins for which folding requires the chaperonin GroEL/GroES (GroE in Escherichia coli, and they revealed that many chaperonin substrates are metabolic enzymes. This result implies the importance of chaperonins in metabolism. However, the relationship between chaperonins and metabolism is still unclear. Results We investigated the distribution of chaperonin substrate enzymes in the metabolic network using network analysis techniques as a first step towards revealing this relationship, and found that as chaperonin requirement increases, substrate enzymes are more laterally distributed in the metabolic. In addition, comparative genome analysis showed that the chaperonin-dependent substrates were less conserved, suggesting that these substrates were acquired later on in evolutionary history. Conclusions This result implies the expansion of metabolic networks due to this chaperonin, and it supports the existing hypothesis of acceleration of evolution by chaperonins. The distribution of chaperonin substrate enzymes in the metabolic network is inexplicable because it does not seem to be associated with individual protein features such as protein abundance, which has been observed characteristically in chaperonin substrates in previous works. However, it becomes clear by considering this expansion process due to chaperonin. This finding provides new insights into metabolic evolution and the roles of chaperonins in living systems.

  7. Probing water density and dynamics in the chaperonin GroEL cavity.

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    Franck, John M; Sokolovski, Miri; Kessler, Naama; Matalon, Erez; Gordon-Grossman, Michal; Han, Song-I; Goldfarb, Daniella; Horovitz, Amnon

    2014-07-02

    ATP-dependent binding of the chaperonin GroEL to its cofactor GroES forms a cavity in which encapsulated substrate proteins can fold in isolation from bulk solution. It has been suggested that folding in the cavity may differ from that in bulk solution owing to steric confinement, interactions with the cavity walls, and differences between the properties of cavity-confined and bulk water. However, experimental data regarding the cavity-confined water are lacking. Here, we report measurements of water density and diffusion dynamics in the vicinity of a spin label attached to a cysteine in the Tyr71 → Cys GroES mutant obtained using two magnetic resonance techniques: electron-spin echo envelope modulation and Overhauser dynamic nuclear polarization. Residue 71 in GroES is fully exposed to bulk water in free GroES and to confined water within the cavity of the GroEL-GroES complex. Our data show that water density and translational dynamics in the vicinity of the label do not change upon complex formation, thus indicating that bulk water-exposed and cavity-confined GroES surface water share similar properties. Interestingly, the diffusion dynamics of water near the GroES surface are found to be unusually fast relative to other protein surfaces studied. The implications of these findings for chaperonin-assisted folding mechanisms are discussed.

  8. Formation of the chaperonin complex studied by 2D NMR spectroscopy

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    Takenaka, Toshio; Nakamura, Takashi; Yanaka, Saeko; Yagi-Utsumi, Maho; Chandak, Mahesh S.; Takahashi, Kazunobu; Paul, Subhankar; Makabe, Koki; Arai, Munehito; Kato, Koichi

    2017-01-01

    We studied the interaction between GroES and a single-ring mutant (SR1) of GroEL by the NMR titration of 15N-labeled GroES with SR1 at three different temperatures (20, 25 and 30°C) in the presence of 3 mM ADP in 100 mM KCl and 10 mM MgCl2 at pH 7.5. We used SR1 instead of wild-type double-ring GroEL to precisely control the stoichiometry of the GroES binding to be 1:1 ([SR1]:[GroES]). Native heptameric GroES was very flexible, showing well resolved cross peaks of the residues in a mobile loop segment (residue 17–34) and at the top of a roof hairpin (Asn51) in the heteronuclear single quantum coherence spectra. The binding of SR1 to GroES caused the cross peaks to disappear simultaneously, and hence it occurred in a single-step cooperative manner with significant immobilization of the whole GroES structure. The binding was thus entropic with a positive entropy change (219 J/mol/K) and a positive enthalpy change (35 kJ/mol), and the binding constant was estimated at 1.9×105 M−1 at 25°C. The NMR titration in 3 mM ATP also indicated that the binding constant between GroES and SR1 increased more than tenfold as compared with the binding constant in 3 mM ADP. These results will be discussed in relation to the structure and mechanisms of the chaperonin GroEL/GroES complex. PMID:29059240

  9. Formation of the chaperonin complex studied by 2D NMR spectroscopy.

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    Toshio Takenaka

    Full Text Available We studied the interaction between GroES and a single-ring mutant (SR1 of GroEL by the NMR titration of 15N-labeled GroES with SR1 at three different temperatures (20, 25 and 30°C in the presence of 3 mM ADP in 100 mM KCl and 10 mM MgCl2 at pH 7.5. We used SR1 instead of wild-type double-ring GroEL to precisely control the stoichiometry of the GroES binding to be 1:1 ([SR1]:[GroES]. Native heptameric GroES was very flexible, showing well resolved cross peaks of the residues in a mobile loop segment (residue 17-34 and at the top of a roof hairpin (Asn51 in the heteronuclear single quantum coherence spectra. The binding of SR1 to GroES caused the cross peaks to disappear simultaneously, and hence it occurred in a single-step cooperative manner with significant immobilization of the whole GroES structure. The binding was thus entropic with a positive entropy change (219 J/mol/K and a positive enthalpy change (35 kJ/mol, and the binding constant was estimated at 1.9×105 M-1 at 25°C. The NMR titration in 3 mM ATP also indicated that the binding constant between GroES and SR1 increased more than tenfold as compared with the binding constant in 3 mM ADP. These results will be discussed in relation to the structure and mechanisms of the chaperonin GroEL/GroES complex.

  10. Chaperonin Structure - The Large Multi-Subunit Protein Complex

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    Irena Roterman

    2009-03-01

    Full Text Available The multi sub-unit protein structure representing the chaperonins group is analyzed with respect to its hydrophobicity distribution. The proteins of this group assist protein folding supported by ATP. The specific axial symmetry GroEL structure (two rings of seven units stacked back to back - 524 aa each and the GroES (single ring of seven units - 97 aa each polypeptide chains are analyzed using the hydrophobicity distribution expressed as excess/deficiency all over the molecule to search for structure-to-function relationships. The empirically observed distribution of hydrophobic residues is confronted with the theoretical one representing the idealized hydrophobic core with hydrophilic residues exposure on the surface. The observed discrepancy between these two distributions seems to be aim-oriented, determining the structure-to-function relation. The hydrophobic force field structure generated by the chaperonin capsule is presented. Its possible influence on substrate folding is suggested.

  11. Structural and mechanistic characterization of an archaeal-like chaperonin from a thermophilic bacterium

    OpenAIRE

    An, Young Jun; Rowland, Sara E.; Na, Jung-Hyun; Spigolon, Dario; Hong, Seung Kon; Yoon, Yeo Joon; Lee, Jung-Hyun; Robb, Frank T.; Cha, Sun-Shin

    2017-01-01

    The chaperonins (CPNs) are megadalton sized hollow complexes with two cavities that open and close to encapsulate non-native proteins. CPNs are assigned to two sequence-related groups that have distinct allosteric mechanisms. In Group I CPNs a detachable co-chaperone, GroES, closes the chambers whereas in Group II a built-in lid closes the chambers. Group I CPNs have a bacterial ancestry, whereas Group II CPNs are archaeal in origin. Here we describe open and closed crystal structures represe...

  12. Chaperonin filaments: The archael cytoskeleton

    Energy Technology Data Exchange (ETDEWEB)

    Trent, J.D.; Kagawa, H.K.; Yaoi, Takuro; Olle, E.; Zaluzec, N.J.

    1997-08-01

    Chaperonins are multi-subunit double-ring complexed composed of 60-kDa proteins that are believed to mediate protein folding in vivo. The chaperonins in the hyperthermophilic archaeon Sulfolobus shibatae are composed of the organism`s two most abundant proteins, which represent 4% of its total protein and have an intracellular concentration of {ge} 3.0 mg/ml. At concentrations of 1.0 mg/ml, purified chaperonin proteins aggregate to form ordered filaments. Filament formation, which requires Mg{sup ++} and nucleotide binding (not hydrolysis), occurs at physiological temperatures under conditions suggesting filaments may exist in vivo. If the estimated 4,600 chaperonins per cell, formed filaments in vivo, they could create a matrix of filaments that would span the diameter of an average S. shibatae cell 100 times. Direct observations of unfixed, minimally treated cells by intermediate voltage electron microscopy (300 kV) revealed an intracellular network of filaments that resembles chaperonin filaments produced in vitro. The hypothesis that the intracellular network contains chaperonins is supported by immunogold analyses. The authors propose that chaperonin activity may be regulated in vivo by filament formation and that chaperonin filaments may serve a cytoskeleton-like function in archaea and perhaps in other prokaryotes.

  13. Differential T-cell recognition of native and recombinant Mycobacterium tuberculosis GroES

    DEFF Research Database (Denmark)

    Rosenkrands, I; Weldingh, K; Ravn, P

    1999-01-01

    Mycobacterium tuberculosis GroES was purified from culture filtrate, and its identity was confirmed by immunoblot analysis and N-terminal sequencing. Comparing the immunological recognition of native and recombinant GroES, we found that whereas native GroES elicited a strong proliferative response...

  14. Chaperonin cofactors, Cpn10 and Cpn20, of green algae and plants function as hetero-oligomeric ring complexes.

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    Tsai, Yi-Chin C; Mueller-Cajar, Oliver; Saschenbrecker, Sandra; Hartl, F Ulrich; Hayer-Hartl, Manajit

    2012-06-08

    The chloroplast chaperonin system of plants and green algae is a curiosity as both the chaperonin cage and its lid are encoded by multiple genes, in contrast to the single genes encoding the two components of the bacterial and mitochondrial systems. In the green alga Chlamydomonas reinhardtii (Cr), three genes encode chaperonin cofactors, with cpn10 encoding a single ∼10-kDa domain and cpn20 and cpn23 encoding tandem cpn10 domains. Here, we characterized the functional interaction of these proteins with the Escherichia coli chaperonin, GroEL, which normally cooperates with GroES, a heptamer of ∼10-kDa subunits. The C. reinhardtii cofactor proteins alone were all unable to assist GroEL-mediated refolding of bacterial ribulose-bisphosphate carboxylase/oxygenase but gained this ability when CrCpn20 and/or CrCpn23 was combined with CrCpn10. Native mass spectrometry indicated the formation of hetero-oligomeric species, consisting of seven ∼10-kDa domains. The cofactor "heptamers" interacted with GroEL and encapsulated substrate protein in a nucleotide-dependent manner. Different hetero-oligomer arrangements, generated by constructing cofactor concatamers, indicated a preferential heptamer configuration for the functional CrCpn10-CrCpn23 complex. Formation of heptamer Cpn10/Cpn20 hetero-oligomers was also observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60α(7)β(7). It appears that hetero-oligomer formation occurs more generally for chloroplast chaperonin cofactors, perhaps adapting the chaperonin system for the folding of specific client proteins.

  15. Expression of a highly toxic protein, Bax, in Escherichia coli by attachment of a leader peptide derived from the GroES cochaperone.

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    Donnelly, M. I.; Stols, L.; Stevens, P. W.; Su, S. X.; Tollaksen, S.; Giometti, C. S.; Joachimiak , A.

    2001-08-01

    Expression of the human apoptosis modulator protein Bax in Escherichia coli is highly toxic, resulting in cell lysis at very low concentrations (Asoh, S., et al., J. Biol. Chem. 273, 11384-11391, 1998). Attempts to express a truncated form of murine Bax in the periplasm by using an expression vector that attached the OmpA signal sequence to the protein failed to alleviate this toxicity. In contrast, attachment of a peptide based on a portion of the E. coli cochaperone GroES reduced Bax's toxicity significantly and allowed good expression. The peptide, which was attached to the N-terminus, included the amino acid sequence of the mobile loop of GroES that has been demonstrated to interact with the chaperonin, GroEL. Under normal growth conditions, expression of this construct was still toxic, but generated a small amount of detectable recombinant Bax. However, when cells were grown in the presence of 2% ethanol, which stimulated overproduction of the molecular chaperones GroEL and DnaK, toxicity was reduced and good overexpression occurred. Two-dimensional gel electrophoresis analysis showed that approximately 15-fold more GroES-loop-Bax was produced under these conditions than under standard conditions and that GroEL and DnaK were elevated approximately 3-fold.

  16. Multivalent binding of nonnative substrate proteins by the chaperonin GroEL.

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    Farr, G W; Furtak, K; Rowland, M B; Ranson, N A; Saibil, H R; Kirchhausen, T; Horwich, A L

    2000-03-03

    The chaperonin GroEL binds nonnative substrate protein in the central cavity of an open ring through exposed hydrophobic residues at the inside aspect of the apical domains and then mediates productive folding upon binding ATP and the cochaperonin GroES. Whether nonnative proteins bind to more than one of the seven apical domains of a GroEL ring is unknown. We have addressed this using rings with various combinations of wild-type and binding-defective mutant apical domains, enabled by their production as single polypeptides. A wild-type extent of binary complex formation with two stringent substrate proteins, malate dehydrogenase or Rubisco, required a minimum of three consecutive binding-proficient apical domains. Rhodanese, a less-stringent substrate, required only two wild-type domains and was insensitive to their arrangement. As a physical correlate, multivalent binding of Rubisco was directly observed in an oxidative cross-linking experiment.

  17. Chaperonin filaments: The archaeal cytoskeleton?

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    Trent, Jonathan D.; Kagawa, Hiromi K.; Yaoi, Takuro; Olle, Eric; Zaluzec, Nestor J.

    1997-01-01

    Chaperonins are high molecular mass double-ring structures composed of 60-kDa protein subunits. In the hyperthermophilic archaeon Sulfolobus shibatae the two chaperonin proteins represent ≈4% of its total protein and have a combined intracellular concentration of >30 mg/ml. At concentrations ≥ 0.5 mg/ml purified chaperonins form filaments in the presence of Mg2+ and nucleotides. Filament formation requires nucleotide binding (not hydrolysis), and occurs at physiological temperatures in biologically relevant buffers, including a buffer made from cell extracts. These observations suggest that chaperonin filaments may exist in vivo and the estimated 4600 chaperonins per cell suggest that such filaments could form an extensive cytostructure. We observed filamentous structures in unfixed, uranyl-acetate-stained S. shibatae cells, which resemble the chaperonin filaments in size and appearance. ImmunoGold (Janssen) labeling using chaperonin antibodies indicated that many chaperonins are associated with insoluble cellular structures and these structures appear to be filamentous in some areas, although they could not be uranyl-acetate-stained. The existence of chaperonin filaments in vivo suggests a mechanism whereby their protein-folding activities can be regulated. More generally, the filaments themselves may play a cytoskeletal role in Archaea. PMID:9144246

  18. Structural and mechanistic characterization of an archaeal-like chaperonin from a thermophilic bacterium.

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    An, Young Jun; Rowland, Sara E; Na, Jung-Hyun; Spigolon, Dario; Hong, Seung Kon; Yoon, Yeo Joon; Lee, Jung-Hyun; Robb, Frank T; Cha, Sun-Shin

    2017-10-10

    The chaperonins (CPNs) are megadalton sized hollow complexes with two cavities that open and close to encapsulate non-native proteins. CPNs are assigned to two sequence-related groups that have distinct allosteric mechanisms. In Group I CPNs a detachable co-chaperone, GroES, closes the chambers whereas in Group II a built-in lid closes the chambers. Group I CPNs have a bacterial ancestry, whereas Group II CPNs are archaeal in origin. Here we describe open and closed crystal structures representing a new phylogenetic branch of CPNs. These Group III CPNs are divergent in sequence and structure from extant CPNs, but are closed by a built-in lid like Group II CPNs. A nucleotide-sensing loop, present in both Group I and Group II CPNs, is notably absent. We identified inter-ring pivot joints that articulate during ring closure. These Group III CPNs likely represent a relic from the ancestral CPN that formed distinct bacterial and archaeal branches.Chaperonins (CPNs) are ATP-dependent protein-folding machines. Here the authors present the open and closed crystal structures of a Group III CPN from the thermophilic bacterium Carboxydothermus hydrogenoformans, discuss its mechanism and structurally compare it with Group I and II CPNs.

  19. Ordered biological nanostructures formed from chaperonin polypeptides

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    Trent, Jonathan D. (Inventor); McMillan, R. Andrew (Inventor); Kagawa, Hiromi (Inventor); Paavola, Chad D. (Inventor)

    2010-01-01

    The following application relates to nanotemplates, nanostructures, nanoarrays and nanodevices formed from wild-type and mutated chaperonin polypeptides, methods of producing such compositions, methods of using such compositions and particular chaperonin polypeptides that can be utilized in producing such compositions.

  20. Folding of newly translated membrane protein CCR5 is assisted by the chaperonin GroEL-GroES

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    Chi, Haixia; Wang, Xiaoqiang; Li, Jiqiang; Ren, Hao; Huang, Fang

    2015-11-01

    The in vitro folding of newly translated human CC chemokine receptor type 5 (CCR5), which belongs to the physiologically important family of G protein-coupled receptors (GPCRs), has been studied in a cell-free system supplemented with the surfactant Brij-35. The freshly synthesized CCR5 can spontaneously fold into its biologically active state but only slowly and inefficiently. However, on addition of the GroEL-GroES molecular chaperone system, the folding of the nascent CCR5 was significantly enhanced, as was the structural stability and functional expression of the soluble form of CCR5. The chaperonin GroEL was partially effective on its own, but for maximum efficiency both the GroEL and its GroES lid were necessary. These results are direct evidence for chaperone-assisted membrane protein folding and therefore demonstrate that GroEL-GroES may be implicated in the folding of membrane proteins.

  1. Chloroplast Chaperonin: An Intricate Protein Folding Machine for Photosynthesis

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    Qian Zhao

    2018-01-01

    Full Text Available Group I chaperonins are large cylindrical-shaped nano-machines that function as a central hub in the protein quality control system in the bacterial cytosol, mitochondria and chloroplasts. In chloroplasts, proteins newly synthesized by chloroplast ribosomes, unfolded by diverse stresses, or translocated from the cytosol run the risk of aberrant folding and aggregation. The chloroplast chaperonin system assists these proteins in folding into their native states. A widely known protein folded by chloroplast chaperonin is the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, an enzyme responsible for the fixation of inorganic CO2 into organic carbohydrates during photosynthesis. Chloroplast chaperonin was initially identified as a Rubisco-binding protein. All photosynthetic eucaryotes genomes encode multiple chaperonin genes which can be divided into α and β subtypes. Unlike the homo-oligomeric chaperonins from bacteria and mitochondria, chloroplast chaperonins are more complex and exists as intricate hetero-oligomers containing both subtypes. The Group I chaperonin requires proper interaction with a detachable lid-like co-chaperonin in the presence of ATP and Mg2+ for substrate encapsulation and conformational transition. Besides the typical Cpn10-like co-chaperonin, a unique co-chaperonin consisting of two tandem Cpn10-like domains joined head-to-tail exists in chloroplasts. Since chloroplasts were proposed as sensors to various environmental stresses, this diversified chloroplast chaperonin system has the potential to adapt to complex conditions by accommodating specific substrates or through regulation at both the transcriptional and post-translational levels. In this review, we discuss recent progress on the unique structure and function of the chloroplast chaperonin system based on model organisms Chlamydomonas reinhardtii and Arabidopsis thaliana. Knowledge of the chloroplast chaperonin system may ultimately lead

  2. Chaperonin GroEL uses asymmetric and symmetric reaction cycles in response to the concentration of non-native substrate proteins.

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    Iizuka, Ryo; Funatsu, Takashi

    2016-01-01

    The Escherichia coli chaperonin GroEL is an essential molecular chaperone that mediates protein folding in association with its cofactor, GroES. It is widely accepted that GroEL alternates the GroES-sealed folding-active rings during the reaction cycle. In other words, an asymmetric GroEL-GroES complex is formed during the cycle, whereas a symmetric GroEL-(GroES)2 complex is not formed. However, this conventional view has been challenged by the recent reports indicating that such symmetric complexes can be formed in the GroEL-GroES reaction cycle. In this review, we discuss the studies of the symmetric GroEL-(GroES)2 complex, focusing on the molecular mechanism underlying its formation. We also suggest that GroEL can be involved in two types of reaction cycles (asymmetric or symmetric) and the type of cycle used depends on the concentration of non-native substrate proteins.

  3. Co-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E. coli-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation

    DEFF Research Database (Denmark)

    Bross, P; Andresen, B S; Winter, V

    1993-01-01

    The influence of co-overexpression of the bacterial chaperonins GroEL and GroES on solubility, tetramer formation and enzyme activity of three variants of heterologously-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) was analysed in order to investigate the molecular mechanism...... and the enzyme activity measured as observed for the wild-type protein. (iii) Solubility of the K304E mutant is in a similar fashion GroESL responsive as the K304Q mutant, but the amount of tetramer observed and the enzyme activity measured do not correlate with the amount of soluble K304E MCAD protein detected...

  4. Substrate protein recognition mechanism of archaeal and eukaryotic chaperonins.

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    Shrestha, Pooja; Jayasinghe, Manori; Stan, George

    2009-03-01

    Chaperonins are double ring-shaped biological nanomachines that assist protein folding. Spectacular conformational changes take place within each chaperonin ring using energy derived from ATP hydrolysis. These changes result in transitions from the open to the closed ring. Substrate proteins bind to the open ring and are encapsulated within the closed ring cavity. We focus on the substrate protein recognition mechanism of archaeal and eukaryotic chaperonins. We predict substrate protein binding sites using structural and bioinformatic analyses of functional states during the chaperonin cycle. Based on large changes in solvent accessible surface area and contact maps we glean the functional role of chaperonin amino acids. During the transition between open to closed chaperonin ring, the largest change in accessible surface area of amino acids is found in helical protrusion and two helices located at the cavity opening. Our calculations suggest that the helical protrusion and two helices constitute the substrate protein binding site.

  5. Dynamics, flexibility, and allostery in molecular chaperonins.

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    Skjærven, Lars; Cuellar, Jorge; Martinez, Aurora; Valpuesta, José María

    2015-09-14

    The chaperonins are a family of molecular chaperones present in all three kingdoms of life. They are classified into Group I and Group II. Group I consists of the bacterial variants (GroEL) and the eukaryotic ones from mitochondria and chloroplasts (Hsp60), while Group II consists of the archaeal (thermosomes) and eukaryotic cytosolic variants (CCT or TRiC). Both groups assemble into a dual ring structure, with each ring providing a protective folding chamber for nascent and denatured proteins. Their functional cycle is powered by ATP binding and hydrolysis, which drives a series of structural rearrangements that enable encapsulation and subsequent release of the substrate protein. Chaperonins have elaborate allosteric mechanisms to regulate their functional cycle. Long-range negative cooperativity between the two rings ensures alternation of the folding chambers. Positive intra-ring cooperativity, which facilitates concerted conformational transitions within the protein subunits of one ring, has only been demonstrated for Group I chaperonins. In this review, we describe our present understanding of the underlying mechanisms and the structure-function relationships in these complex protein systems with a particular focus on the structural dynamics, allostery, and associated conformational rearrangements. Copyright © 2015. Published by Elsevier B.V.

  6. Ordered nanoparticle arrays formed on engineered chaperonin protein templates

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    McMillan, R. Andrew; Paavola, Chad D.; Howard, Jeanie; Chan, Suzanne L.; Zaluzec, Nestor J.; Trent, Jonathan D.

    2002-01-01

    Traditional methods for fabricating nanoscale arrays are usually based on lithographic techniques. Alternative new approaches rely on the use of nanoscale templates made of synthetic or biological materials. Some proteins, for example, have been used to form ordered two-dimensional arrays. Here, we fabricated nanoscale ordered arrays of metal and semiconductor quantum dots by binding preformed nanoparticles onto crystalline protein templates made from genetically engineered hollow double-ring structures called chaperonins. Using structural information as a guide, a thermostable recombinant chaperonin subunit was modified to assemble into chaperonins with either 3 nm or 9 nm apical pores surrounded by chemically reactive thiols. These engineered chaperonins were crystallized into two-dimensional templates up to 20 microm in diameter. The periodic solvent-exposed thiols within these crystalline templates were used to size-selectively bind and organize either gold (1.4, 5 or 10nm) or CdSe-ZnS semiconductor (4.5 nm) quantum dots into arrays. The order within the arrays was defined by the lattice of the underlying protein crystal. By combining the self-assembling properties of chaperonins with mutations guided by structural modelling, we demonstrate that quantum dots can be manipulated using modified chaperonins and organized into arrays for use in next-generation electronic and photonic devices.

  7. CHAPERONIN 20 mediates iron superoxide dismutase (FeSOD) activity independent of its co-chaperonin role in Arabidopsis chloroplasts.

    Science.gov (United States)

    Kuo, W Y; Huang, C H; Liu, A C; Cheng, C P; Li, S H; Chang, W C; Weiss, C; Azem, A; Jinn, T L

    2013-01-01

    Iron superoxide dismutases (FeSODs; FSDs) are primary antioxidant enzymes in Arabidopsis thaliana chloroplasts. The stromal FSD1 conferred the only detectable FeSOD activity, whereas the thylakoid membrane- and nucleoid-co-localized FSD2 and FSD3 double mutant showed arrested chloroplast development. FeSOD requires cofactor Fe for its activity, but its mechanism of activation is unclear. We used reversed-phase high-performance liquid chromatography (HPLC), gel filtration chromatography, LC-MS/MS, protoplast transient expression and virus-induced gene silencing (VIGS) analyses to identify and characterize a factor involved in FeSOD activation. We identified the chloroplast-localized co-chaperonin CHAPERONIN 20 (CPN20) as a mediator of FeSOD activation by direct interaction. The relationship between CPN20 and FeSOD was confirmed by in vitro experiments showing that CPN20 alone could enhance FSD1, FSD2 and FSD3 activity. The in vivo results showed that CPN20-overexpressing mutants and mutants with defective co-chaperonin activity increased FSD1 activity, without changing the chaperonin CPN60 protein level, and VIGS-induced downregulation of CPN20 also led to decreased FeSOD activity. Our findings reveal that CPN20 can mediate FeSOD activation in chloroplasts, a role independent of its known function in the chaperonin system. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  8. Chaperonin filaments : their formation and an evaluation of methods for studying them.

    Energy Technology Data Exchange (ETDEWEB)

    Yaoi, T.; Kagawa, K. H.; Trent, J. D.; Center for Mechanistic Biology and Biotechnology

    1998-08-01

    Chaperonins are multisubunit protein complexes that can be isolated from cells as high-molecular-weight structures that appear as double rings in the electron microscope. We recently discovered that chaperonin double rings isolated from the hyperthermophilic archaeon Sulfolobus shibatae, when incubated at physiological temperatures in the presence of ATP and Mg{sup 2+}, stacked into filaments; we hypothesized that these filaments are related to filaments seen inside S. shibatae cells and that chaperonins exist as filaments in vivo. This paper elucidates the conditions under which we have observed S. shibatae chaperonins to form filaments and evaluates native polyacrylamide gel electrophoresis (PAGE), TEM, spectrophotometry, and centrifugation as methods for studying these filaments. We observed that in the presence of Mg{sup 2+} combined with ATP, ADP, ATP{gamma}S, or GTP, native PAGE indicated that chaperonin subunits assembled into double rings and that the conformation of these double rings was effected by nucleotide binding, but we saw no indication of chaperonin filament formation. Under these same conditions, however, TEM, spectroscopy, and centrifugation methods indicated that chaperonin subunits and double rings had assembled into filaments. We determined that this discrepancy in the representation of the chaperonin structure was due to the native PAGE method itself. When we exposed chaperonin filaments to the electrophoretic field used in native PAGE, the filaments dissociated into double rings. This suggests that TEM, spectrophotometry, and centrifugation are the preferred methods for studying the higher-order structures of chaperonins, which are likely to be of biological significance.

  9. Versatile platform for nanotechnology based on circular permutations of chaperonin protein

    Science.gov (United States)

    Paavola, Chad D. (Inventor); Trent, Jonathan D. (Inventor); Chan, Suzanne L. (Inventor); Li, Yi-Fen (Inventor); McMillan, R. Andrew (Inventor); Kagawa, Hiromi (Inventor)

    2010-01-01

    The present invention provides chaperonin polypeptides which are modified to include N-terminal and C-terminal ends that are relocated from the central pore region to various different positions in the polypeptide which are located on the exterior of the folded modified chaperonin polypeptide. In the modified chaperonin polypeptide, the naturally-occurring N-terminal and C-terminal ends are joined together directly or with an intervening linker peptide sequence. The relocated N-terminal or C-terminal ends can be covalently joined to, or bound with another molecule such as a nucleic acid molecule, a lipid, a carbohydrate, a second polypeptide, or a nanoparticle. The modified chaperonin polypeptides can assemble into double-ringed chaperonin structures. Further, the chaperonin structures can organize into higher order structures such as nanofilaments or nanoarrays which can be used to produce nanodevices and nanocoatings.

  10. Novel chaperonins are prevalent in the virioplankton and demonstrate links to viral biology and ecology

    Science.gov (United States)

    Marine, Rachel L; Nasko, Daniel J; Wray, Jeffrey; Polson, Shawn W; Wommack, K Eric

    2017-01-01

    Chaperonins are protein-folding machinery found in all cellular life. Chaperonin genes have been documented within a few viruses, yet, surprisingly, analysis of metagenome sequence data indicated that chaperonin-carrying viruses are common and geographically widespread in marine ecosystems. Also unexpected was the discovery of viral chaperonin sequences related to thermosome proteins of archaea, indicating the presence of virioplankton populations infecting marine archaeal hosts. Virioplankton large subunit chaperonin sequences (GroELs) were divergent from bacterial sequences, indicating that viruses have carried this gene over long evolutionary time. Analysis of viral metagenome contigs indicated that: the order of large and small subunit genes was linked to the phylogeny of GroEL; both lytic and temperate phages may carry group I chaperonin genes; and viruses carrying a GroEL gene likely have large double-stranded DNA (dsDNA) genomes (>70 kb). Given these connections, it is likely that chaperonins are critical to the biology and ecology of virioplankton populations that carry these genes. Moreover, these discoveries raise the intriguing possibility that viral chaperonins may more broadly alter the structure and function of viral and cellular proteins in infected host cells. PMID:28731469

  11. Novel chaperonins are prevalent in the virioplankton and demonstrate links to viral biology and ecology.

    Science.gov (United States)

    Marine, Rachel L; Nasko, Daniel J; Wray, Jeffrey; Polson, Shawn W; Wommack, K Eric

    2017-11-01

    Chaperonins are protein-folding machinery found in all cellular life. Chaperonin genes have been documented within a few viruses, yet, surprisingly, analysis of metagenome sequence data indicated that chaperonin-carrying viruses are common and geographically widespread in marine ecosystems. Also unexpected was the discovery of viral chaperonin sequences related to thermosome proteins of archaea, indicating the presence of virioplankton populations infecting marine archaeal hosts. Virioplankton large subunit chaperonin sequences (GroELs) were divergent from bacterial sequences, indicating that viruses have carried this gene over long evolutionary time. Analysis of viral metagenome contigs indicated that: the order of large and small subunit genes was linked to the phylogeny of GroEL; both lytic and temperate phages may carry group I chaperonin genes; and viruses carrying a GroEL gene likely have large double-stranded DNA (dsDNA) genomes (>70 kb). Given these connections, it is likely that chaperonins are critical to the biology and ecology of virioplankton populations that carry these genes. Moreover, these discoveries raise the intriguing possibility that viral chaperonins may more broadly alter the structure and function of viral and cellular proteins in infected host cells.

  12. Chaperonin filaments: their formation and an evaluation of methods for studying them.

    Science.gov (United States)

    Yaoi, T; Kagawa, H K; Trent, J D

    1998-08-01

    Chaperonins are multisubunit protein complexes that can be isolated from cells as high-molecular-weight structures that appear as double rings in the electron microscope. We recently discovered that chaperonin double rings isolated from the hyperthermophilic archaeon Sulfolobus shibatae, when incubated at physiological temperatures in the presence of ATP and Mg2+, stacked into filaments; we hypothesized that these filaments are related to filaments seen inside S. shibatae cells and that chaperonins exist as filaments in vivo (J. D. Trent et al., 1997, Proc. Natl. Acad. Sci. USA 94, 5383-5388). This paper elucidates the conditions under which we have observed S. shibatae chaperonins to form filaments and evaluates native polyacrylamide gel electrophoresis (PAGE), TEM, spectrophotometry, and centrifugation as methods for studying these filaments. We observed that in the presence of Mg2+ combined with ATP, ADP, ATPgammaS, or GTP, native PAGE indicated that chaperonin subunits assembled into double rings and that the conformation of these double rings was effected by nucleotide binding, but we saw no indication of chaperonin filament formation. Under these same conditions, however, TEM, spectroscopy, and centrifugation methods indicated that chaperonin subunits and double rings had assembled into filaments. We determined that this discrepancy in the representation of the chaperonin structure was due to the native PAGE method itself. When we exposed chaperonin filaments to the electrophoretic field used in native PAGE, the filaments dissociated into double rings. This suggests that TEM, spectrophotometry, and centrifugation are the preferred methods for studying the higher-order structures of chaperonins, which are likely to be of biological significance. Copyright 1998 Academic Press.

  13. Dataset concerning GroEL chaperonin interaction with proteins

    Directory of Open Access Journals (Sweden)

    V.V. Marchenkov

    2016-03-01

    Full Text Available GroEL chaperonin is well-known to interact with a wide variety of polypeptide chains. Here we show the data related to our previous work (http://dx.doi.org/10.1016/j.pep.2015.11.020 [1], and concerning the interaction of GroEL with native (lysozyme, α-lactalbumin and denatured (lysozyme, α-lactalbumin and pepsin proteins in solution. The use of affinity chromatography on the base of denatured pepsin for GroEL purification from fluorescent impurities is represented as well.

  14. Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis

    DEFF Research Database (Denmark)

    Kilstrup, Mogens; Jacobsen, Susanne; Hammer, Karin

    1997-01-01

    pattern. The fast-induced proteins (including DnaK) showed an abruptly increased rate of synthesis during the first 10 min, declining to intermediate levels after 15 min, GroEL and GroES, which also belong to this group, maintained a high rate of synthesis after 15 min, The class of slowly induced......, by comparison of prototrophic wild-type strains and auxotrophic domesticated (daily) strains. In a study of the capacity of domesticated strains to perform directed responses toward various stress conditions, we have analyzed the heat and salt stress response in the established L,. lactis subsp. cremoris...... laboratory strain MG1363, which was originally derived from a dairy strain, After two-dimensional separation of proteins, the DnaK, GroEL, and GroES heat shock proteins, the HrcA (Orf1) heat shack repressor, and the glycolytic enzymes pyruvate kinase, glyceral-dehyde-3-phosphate dehydrogenase...

  15. Chaperonins fight aminoglycoside-induced protein misfolding and promote short-term tolerance in Escherichia coli

    DEFF Research Database (Denmark)

    Goltermann, Lise; Good, Liam; Bentin, Thomas

    2013-01-01

    For almost half of a century, we have known that aminoglycoside antibiotics corrupt ribosomes, causing translational misreading, yet it remains unclear whether or not misreading triggers protein misfolding, and possible effects of chaperone action on drug susceptibilities are poorly understood....... Here, we show that aminoglycosides cause cytosolic protein misfolding and that chaperonin GroEL/GroES overexpression counters this defect. During aminoglycoside exposure to exponential cultures, chaperonin overexpression protected the bacterial membrane potential, rescued cell growth, and facilitated...

  16. Isolation and characterization of a Paracentrotus lividus cDNA encoding a stress-inducible chaperonin

    Science.gov (United States)

    Gianguzza, Fabrizio; Antonietta Ragusa, Maria; Roccheri, Maria Carmela; Liegro, Italia Di; Rinaldi, Anna Maria

    2000-01-01

    Chaperonins are ubiquitous proteins that facilitate protein folding in an adenosine triphosphate–dependent manner. Here we report the isolation of a sea urchin cDNA (Plhsp60) coding for mitochondrial chaperonin (Cpn60), whose basal expression is further enhanced by heat shock. The described cDNA corresponds to a full-length mRNA encoding a protein of 582 amino acids, the first 32 of which constitute a putative mitochondrial targeting leader sequence. Comparative analysis has demonstrated that this protein is highly conserved in evolution. PMID:11147969

  17. Chaperonin of Group I: Oligomeric Spectrum and Biochemical and Biological Implications

    Directory of Open Access Journals (Sweden)

    Silvia Vilasi

    2018-01-01

    Full Text Available Chaperonins play various physiological roles and can also be pathogenic. Elucidation of their structure, e.g., oligomeric status and post-translational modifications (PTM, is necessary to understand their functions and mechanisms of action in health and disease. Group I chaperonins form tetradecamers with two stacked heptameric rings. The tetradecamer is considered the typical functional complex for folding of client polypeptides. However, other forms such as the monomer and oligomers with smaller number of subunits than the classical tetradecamer, also occur in cells. The properties and functions of the monomer and oligomers, and their roles in chaperonin-associated diseases are still incompletely understood. Chaperonin I in eukaryotes occurs in various locations, not just the mitochondrion, which is its canonical place of residence and function. Eukaryotic Chaperonin I, namely Hsp60 (designated HSP60 or HSPD1 in humans has, indeed, been found in the cytosol; the plasma-cell membrane; on the outer surface of cells; in the intercellular space; in biological liquids such as lymph, blood, and cerebrospinal fluid; and in secretions, for instance saliva and urine. Hsp60 has also been found in cell-derived vesicles such as exosomes. The functions of Hsp60 in all these non-canonical locales are still poorly characterized and one of the questions not yet answered is in what form, i.e., monomer or oligomer, is the chaperonin present in these non-canonical locations. In view of the steady increase in interest on chaperonopathies over the last several years, we have studied human HSP60 to determine its role in various diseases, its locations in cells and tissues and migrations in the body, and its post-translational modifications that might have an impact on its location and function. We also carried out experiments to characterize the oligomeric status of extramitochondrial of HSP60 in solution. Here, we provide an overview of our results, focusing on

  18. Single-molecule fluorescence polarization study of conformational change in archaeal group II chaperonin.

    Directory of Open Access Journals (Sweden)

    Ryo Iizuka

    Full Text Available Group II chaperonins found in archaea and in eukaryotic cytosol mediate protein folding without a GroES-like cofactor. The function of the cofactor is substituted by the helical protrusion at the tip of the apical domain, which forms a built-in lid on the central cavity. Although many studies on the change in lid conformation coupled to the binding and hydrolysis of nucleotides have been conducted, the molecular mechanism of lid closure remains poorly understood. Here, we performed a single-molecule polarization modulation to probe the rotation of the helical protrusion of a chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1. We detected approximately 35° rotation of the helical protrusion immediately after photorelease of ATP. The result suggests that the conformational change from the open lid to the closed lid state is responsible for the approximately 35° rotation of the helical protrusion.

  19. Circular Permutation of a Chaperonin Protein: Biophysics and Application to Nanotechnology

    Science.gov (United States)

    Paavola, Chad; Chan, Suzanne; Li, Yi-Fen; McMillan, R. Andrew; Trent, Jonathan

    2004-01-01

    We have designed five circular permutants of a chaperonin protein derived from the hyperthermophilic organism Sulfolobus shibatae. These permuted proteins were expressed in E. coli and are well-folded. Furthermore, all the permutants assemble into 18-mer double rings of the same form as the wild-type protein. We characterized the thermodynamics of folding for each permutant by both guanidine denaturation and differential scanning calorimetry. We also examined the assembly of chaperonin rings into higher order structures that may be used as nanoscale templates. The results show that circular permutation can be used to tune the thermodynamic properties of a protein template as well as facilitating the fusion of peptides, binding proteins or enzymes onto nanostructured templates.

  20. The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT.

    Science.gov (United States)

    Joachimiak, Lukasz A; Walzthoeni, Thomas; Liu, Corey W; Aebersold, Ruedi; Frydman, Judith

    2014-11-20

    The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical, and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate-binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to TRiC's unique ability to fold obligate substrates.

  1. Conformational states of ribulosebisphosphate carboxylase and their interaction with chaperonin 60.

    Science.gov (United States)

    van der Vies, S M; Viitanen, P V; Gatenby, A A; Lorimer, G H; Jaenicke, R

    1992-04-14

    fallen to the CAC. In the presence of a molar excess of chaperonin 60 oligomers, the I1 state forms a stable binary complex. No stable binary complex between chaperonin 60 and the N1 state could be detected. Formation of the chaperonin 60-I1 binary complex arrests the spontaneous folding process. The I1 state becomes resistant to interaction with chaperonin 60 with kinetics indistinguishable from those associated with the appearance of the native states. In vitro complementation analysis indicated that the product of the chaperonin-facilitated process is monomeric.(ABSTRACT TRUNCATED AT 400 WORDS)

  2. GeneCAT--novel webtools that combine BLAST and co-expression analyses

    DEFF Research Database (Denmark)

    Mutwil, Marek; Obro, Jens; Willats, William G T

    2008-01-01

    The gene co-expression analysis toolbox (GeneCAT) introduces several novel microarray data analyzing tools. First, the multigene co-expression analysis, combined with co-expressed gene networks, provides a more powerful data mining technique than standard, single-gene co-expression analysis. Second...... orthologs in the plant model organisms Arabidopsis thaliana and Hordeum vulgare (Barley). GeneCAT is equipped with expression data for the model plant A. thaliana, and first to introduce co-expression mining tools for the monocot Barley. GeneCAT is available at http://genecat.mpg.de....

  3. Symmetric GroEL:GroES2 complexes are the protein-folding functional form of the chaperonin nanomachine.

    Science.gov (United States)

    Yang, Dong; Ye, Xiang; Lorimer, George H

    2013-11-12

    Using calibrated FRET, we show that the simultaneous occupancy of both rings of GroEL by ATP and GroES occurs, leading to the rapid formation of symmetric GroEL:GroES2 "football" particles regardless of the presence or absence of substrate protein (SP). In the absence of SP, these symmetric particles revert to asymmetric GroEL:GroES1 "bullet" particles. The breakage of GroES symmetry requires the stochastic hydrolysis of ATP and the breakage of nucleotide symmetry. These asymmetric particles are both persistent and dynamic; they turnover via the asymmetric cycle. When challenged with SP, however, they revert to symmetric particles within a second. In the presence of SP, the symmetric particles are also persistent and dynamic. They turn over via the symmetric cycle. Under these conditions, the stochastic hydrolysis of ATP and the breakage of nucleotide symmetry also occur within the ensemble of particles. However, on account of SP-catalyzed ADP/ATP exchange, GroES symmetry is rapidly restored. The residence time of both GroES and SP on functional GroEL is reduced to ∼1 s, enabling many more iterations than was previously believed possible, consistent with the iterative annealing mechanism. This result is inconsistent with currently accepted models. Using a foldable SP, we show that as the SP folds to the native state and the population of unfolded SP declines, the population of symmetric particles reverts to asymmetric particles in parallel, a result that is consistent with the former being the folding functional form.

  4. Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression

    Directory of Open Access Journals (Sweden)

    Neubauer Peter

    2011-08-01

    Full Text Available Abstract Background Eukaryotic Ribonuclease inhibitor (RI, belonging to the RNH1 family, is distinguished by unique features - a high sensitivity to oxidation due to the large number of reduced cysteins and a high hydrophobicity, which made most production approaches so far unsuccessful or resulted in very low yields. In this work efficient in vivo folding of native RI in the Escherichia coli cytoplasm was obtained by external addition of a reducing agent in tandem with oxygen limitation and overproduction of a molecular chaperonin. After optimisation of the production conditions in the shake flask scale the process was scaled up to high cell densities by applying a glucose limited fed-batch procedure. Results RI production in a T7 RNA polymerase based system results in accumulation of aggregated inactive product in inclusion bodies. Combination of addition of the reductant DTT, low production temperature and coexpression of the chaperonin GroELS resulted in high level production of approximately 25 mg g-1 CDW active RI in E. coli ER2566 pET21b, corresponding to approximately 800 kU g-1 cell wet weight. Further conditional screening under fed-batch-like conditions with the EnBase® technology and scale up into the bioreactor scale resulted in an efficient high cell density glucose and oxygen limited fed-batch process with a final cell dry weight of 25 g L-1 and a total RI yield of app. 625 mg L-1 (volumetric activity of 80,000 kU L-1. The E. coli based production constructs showed a very high robustness. The recombinant culture maintained its productivity despite the combination of the toxic growth conditions, the substrate limited production mode in tandem with a high level expression of several recombinant proteins, the set of molecular chaperonins and the target protein (RI. Conclusions High level production of active RI in E. coli in a T7 RNA polymerase expression system depends on the following factors: (i addition of a reducing agent, (ii

  5. Properties of the alpha subunit of a Chaperonin from the hyperthermophilic Crenarchaeon Aeropyrum pernix K1.

    Science.gov (United States)

    Son, Hae-Jin; Shin, Eun-Jung; Nam, Soo-Wan; Kim, Dong-Eun; Jeon, Sung-Jong

    2007-01-01

    The gene encoding for a putative thermosome from the hyperthermophilic crenarchaeon Aeropyrum pernix K1 (ApcpnA) was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (accession no. APE0907) from A. pernix K1 showed some homology with other group II chaperonins from archaea. The recombinant ApcpnA protein has a molecular mass of 60 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and exhibited ATPase activity with an optimum temperature and pH of 90 degrees C and 5.0, respectively. The ATPase activity was found to be dependent on manganese and potassium ions, but not magnesium ion. The K(m) for ATP at pH 5.0 and 90 degrees C was 10.04 (+/- 1.31) microM, and k(cat) was determined to be 2.21 (+/- 0.11) min(-1) for the ApcpnA monomer. The recombinant ApcpnA prevents thermal aggregation of bovine rhodanese and enhances the thermal stability of alcohol dehydrogenase in vitro, indicating that the protein is suitable as a molecular chaperonin in the high-temperature environment.

  6. Knotting and unknotting proteins in the chaperonin cage: Effects of the excluded volume.

    Directory of Open Access Journals (Sweden)

    Szymon Niewieczerzal

    Full Text Available Molecular dynamics simulations are used to explore the effects of chaperonin-like cages on knotted proteins with very low sequence similarity, different depths of a knot but with a similar fold, and the same type of topology. The investigated proteins are VirC2, DndE and MJ0366 with two depths of a knot. A comprehensive picture how encapsulation influences folding rates is provided based on the analysis of different cage sizes and temperature conditions. Neither of these two effects with regard to knotted proteins has been studied by means of molecular dynamics simulations with coarse-grained structure-based models before. We show that encapsulation in a chaperonin is sufficient to self-tie and untie small knotted proteins (VirC2, DndE, for which the equilibrium process is not accessible in the bulk solvent. Furthermore, we find that encapsulation reduces backtracking that arises from the destabilisation of nucleation sites, smoothing the free energy landscape. However, this effect can also be coupled with temperature rise. Encapsulation facilitates knotting at the early stage of folding and can enhance an alternative folding route. Comparison to unknotted proteins with the same fold shows directly how encapsulation influences the free energy landscape. In addition, we find that as the size of the cage decreases, folding times increase almost exponentially in a certain range of cage sizes, in accordance with confinement theory and experimental data for unknotted proteins.

  7. Chaperonin GroEL/GroES over-expression promotes multi-drug resistance in E. coli following exposure to aminoglycoside antibiotics

    Directory of Open Access Journals (Sweden)

    Lise eGoltermann

    2016-01-01

    Full Text Available Antibiotic resistance is an increasing challenge to modern healthcare. Aminoglycoside antiobiotics cause translation corruption and protein misfolding and aggregation in Escherichia coli. We previously showed that chaperonin GroEL/GroES depletion and overexpression sensitize and promote short-term tolerance, respectively, to this drug class. Here we show that chaperonin GroEL/GroES over-expression accelerates acquisition of aminoglycoside resistance and multi-drug resistance following sub-lethal aminoglycoside antibiotic exposure. Chaperonin buffering could provide a novel mechanism for antibiotic resistance and multi-drug resistance development.

  8. Characterization of differentially expressed genes using high-dimensional co-expression networks

    DEFF Research Database (Denmark)

    Coelho Goncalves de Abreu, Gabriel; Labouriau, Rodrigo S.

    2010-01-01

    We present a technique to characterize differentially expressed genes in terms of their position in a high-dimensional co-expression network. The set-up of Gaussian graphical models is used to construct representations of the co-expression network in such a way that redundancy and the propagation...

  9. Hi-C Chromatin Interaction Networks Predict Co-expression in the Mouse Cortex

    NARCIS (Netherlands)

    Babaei, S.; Mahfouz, A.M.E.T.A.; Hulsman, M.; Lelieveldt, B.P.F.; De Ridder, J.; Reinders, M.J.T.

    2015-01-01

    The three dimensional conformation of the genome in the cell nucleus influences important biological processes such as gene expression regulation. Recent studies have shown a strong correlation between chromatin interactions and gene co-expression. However, predicting gene co-expression from

  10. Inter-species differences of co-expression of neighboring genes in eukaryotic genomes

    Directory of Open Access Journals (Sweden)

    Inaoka Hidenori

    2004-01-01

    Full Text Available Abstract Background There is increasing evidence that gene order within the eukaryotic genome is not random. In yeast and worm, adjacent or neighboring genes tend to be co-expressed. Clustering of co-expressed genes has been found in humans, worm and fruit flies. However, in mice and rats, an effect of chromosomal distance (CD on co-expression has not been investigated yet. Also, no cross-species comparison has been made so far. We analyzed the effect of CD as well as normalized distance (ND using expression data in six eukaryotic species: yeast, fruit fly, worm, rat, mouse and human. Results We analyzed 24 sets of expression data from the six species. Highly co-expressed pairs were sorted into bins of equal sized intervals of CD, and a co-expression rate (CoER in each bin was calculated. In all datasets, a higher CoER was obtained in a short CD range than a long distance range. These results show that across all studied species, there was a consistent effect of CD on co-expression. However, the results using the ND show more diversity. Intra- and inter-species comparisons of CoER reveal that there are significant differences in the co-expression rates of neighboring genes among the species. A pair-wise BLAST analysis finds 8 – 30 % of the highly co-expressed pairs are duplic ated genes. Conclusion We confirmed that in the six eukaryotic species, there was a consistent tendency that neighboring genes are likely to be co-expressed. Results of pair-wised BLAST indicate a significant effect of non-duplicated pairs on co-expression. A comparison of CD and ND suggests the dominant effect of CD.

  11. Differentially correlated genes in co-expression networks control phenotype transitions.

    Science.gov (United States)

    Thomas, Lina D; Vyshenska, Dariia; Shulzhenko, Natalia; Yambartsev, Anatoly; Morgun, Andrey

    2016-01-01

    Co-expression networks are a tool widely used for analysis of "Big Data" in biology that can range from transcriptomes to proteomes, metabolomes and more recently even microbiomes. Several methods were proposed to answer biological questions interrogating these networks. Differential co-expression analysis is a recent approach that measures how gene interactions change when a biological system transitions from one state to another. Although the importance of differentially co-expressed genes to identify dysregulated pathways has been noted, their role in gene regulation is not well studied. Herein we investigated differentially co-expressed genes in a relatively simple mono-causal process (B lymphocyte deficiency) and in a complex multi-causal system (cervical cancer). Co-expression networks of B cell deficiency (Control and BcKO) were reconstructed using Pearson correlation coefficient for two mus musculus datasets: B10.A strain (12 normal, 12 BcKO) and BALB/c strain (10 normal, 10 BcKO). Co-expression networks of cervical cancer (normal and cancer) were reconstructed using local partial correlation method for five datasets (total of 64 normal, 148 cancer). Differentially correlated pairs were identified along with the location of their genes in BcKO and in cancer networks. Minimum Shortest Path and Bi-partite Betweenness Centrality where statistically evaluated for differentially co-expressed genes in corresponding networks.    Results: We show that in B cell deficiency the differentially co-expressed genes are highly enriched with immunoglobulin genes (causal genes). In cancer we found that differentially co-expressed genes act as "bottlenecks" rather than causal drivers with most flows that come from the key driver genes to the peripheral genes passing through differentially co-expressed genes. Using in vitro knockdown experiments for two out of 14 differentially co-expressed genes found in cervical cancer (FGFR2 and CACYBP), we showed that they play

  12. Co-Expression of Neighboring Genes in the Zebrafish (Danio rerio Genome

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    Daryi Wang

    2009-08-01

    Full Text Available Neighboring genes in the eukaryotic genome have a tendency to express concurrently, and the proximity of two adjacent genes is often considered a possible explanation for their co-expression behavior. However, the actual contribution of the physical distance between two genes to their co-expression behavior has yet to be defined. To further investigate this issue, we studied the co-expression of neighboring genes in zebrafish, which has a compact genome and has experienced a whole genome duplication event. Our analysis shows that the proportion of highly co-expressed neighboring pairs (Pearson’s correlation coefficient R>0.7 is low (0.24% ~ 0.67%; however, it is still significantly higher than that of random pairs. In particular, the statistical result implies that the co-expression tendency of neighboring pairs is negatively correlated with their physical distance. Our findings therefore suggest that physical distance may play an important role in the co-expression of neighboring genes. Possible mechanisms related to the neighboring genes’ co-expression are also discussed.

  13. Potential of mean force between a large solute and a biomolecular complex: A model analysis on protein flux through chaperonin system

    Science.gov (United States)

    Amano, Ken-ich; Oshima, Hiraku; Kinoshita, Masahiro

    2011-11-01

    Insertion of a large solute into an even larger vessel comprising biopolymers followed by release of the same solute from it is one of the important functions sustaining life. As a typical example, an unfolded protein is inserted into a chaperonin from bulk aqueous solution, a cochaperonin acting as a lid is attached to the chaperonin rim and the protein folds into its native structure within the closed cavity, the cochaperonin is detached after the folding is finished, and the folded protein is released back to the bulk solution. On the basis of the experimental observations manifesting that the basic aspects of the protein flux through the chaperonin system is independent of the chaperonin, cochaperonin, and protein species, we adopt a simple model system with which we can cover the whole cycle of the protein flux. We calculate the spatial distribution of the solvent-mediated potential of mean force (PMF) between a spherical solute and a cylindrical vessel or vessel/lid complex. The calculation is performed using the three-dimensional integral equation theory, and the PMF is decomposed into energetic and entropic components. We argue that an unfolded protein with a larger excluded volume (EV) and weak hydrophobicity is entropically inserted into the chaperonin cavity and constrained within a small space almost in its center. The switch from insertion to release is achieved by decreasing the EV and turning the protein surface hydrophilic in the folding process. For this release, in which the energetic component is a requisite, the feature that the chaperonin inner surface in the absence of the cochaperonin is not hydrophilic plays essential roles. On the other hand, the inner surface of the chaperonin/cochaperonin complex is hydrophilic, and the protein is energetically repelled from it: The protein remains constrained within the small space mentioned above without contacting the inner surface for correct folding. The structural and inner-surface properties of the

  14. Cooperativity of alpha- and beta-subunits of group II chaperonin from the hyperthermophilic archaeum Aeropyrum pernix K1.

    Science.gov (United States)

    Kim, Jeong-Hwan; Lee, Jin-Woo; Shin, Eun-Jung; Nam, Soo-Wan

    2011-02-01

    alpha- and beta-subunits (ApCpnA and ApCpnB) are group II chaperonins from the hyperthermophilic archaeum Aeropyrum pernix K1, specialized in preventing the aggregation and inactivation of substrate proteins under conditions of transient heat stress. In the present study, the cooperativity of alpha- and beta-subunits from the A. pernix K1 was investigated. The ApCpnA and ApCpnB chaperonin genes were overexpressed in E. coli Rosetta and Codonplus (DE3), respectively. Each of the recombinant alpha- and beta- subunits was purified to 92% and 94% by using anionexchange chromatography. The cooperative activity between purified alpha- and beta-subunits was examined using citrate synthase (CS), alcohol dehydrogenase (ADH), and malate dehydrogenase (MDH) as substrate proteins. The addition of both alpha- and beta-subunits could effectively protect CS and ADH from thermal aggregation and inactivation at 43 degreesC and 50 degreesC, respectively, and MDH from thermal inactivation at 80 degreesC and 85 degreesC. Moreover, in the presence of ATP, the protective effects of alpha- and beta-subunits on CS from thermal aggregation and inactivation, and ADH from thermal aggregation, were more enhanced, whereas cooperation between chaperonins and ATP in protection activity on ADH and MDH (at 85 degreesC) from thermal inactivation was not observed. Specifically, the presence of both alpha- and beta- subunits could effectively protect MDH from thermal inactivation at 80 degreesC in an ATP-dependent manner.

  15. A series of bacterial co-expression vectors with rare-cutter recognition sequences.

    Science.gov (United States)

    Wakamori, Masatoshi; Umehara, Takashi; Yokoyama, Shigeyuki

    2010-11-01

    The bacterial co-expression method is a useful protein expression technique to reconstitute a hetero-oligomeric protein complex in vitro. However, during the plasmid subcloning for co-expression, unintended cleavage at the sequences of target cDNAs becomes more frequent as the number of DNA inserts increases. This problem also makes it difficult to change the combination of targeted proteins after preparing a certain co-expression construct. To avoid this problem, we have developed a series of bacterial co-expression vectors, in which each translation cassette can be subcloned at a set of rare-cutter restriction enzyme sites. We selected 9 different rare-cutter restriction enzymes that recognize a 7 or 8-base-pair sequence, and constructed 27 kinds of cloning vectors and 3 kinds of co-expression vectors, utilizing the rare-cutter recognition sequences as multi-cloning sites. Using this vector system, we co-expressed and co-purified a 7-subunit protein complex composed of the mammalian 26S proteasome regulatory subunits RPT1 to RPT6, and their associated factor, gankyrin. We verified the presence of all 7 subunits by western blotting, by taking advantage of the vector system in which the target proteins can be fused with a broad repertoire of epitope tags. Copyright 2010 Elsevier Inc. All rights reserved.

  16. Pathway enrichment and co-expression cluster analysis - FANTOM5 | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us FANTOM5 Pathway enrichment and co-expression cluster analysis Data detail Data name Pathway ...enrichment and co-expression cluster analysis DOI 10.18908/lsdba.nbdc01389-003.V002 Version V2 10.18908/lsdb...nrichment and co-expression cluster analysis Data file File name: Co-expression_c.../ File size: 86 MB Simple search URL - Data acquisition method - Data analysis method Co-expression cluster analysis... Gostat enrichment analysis Pathway enrichment analysis Number of data en

  17. Allosteric transitions of supramolecular systems explored by network models: application to chaperonin GroEL.

    Directory of Open Access Journals (Sweden)

    Zheng Yang

    2009-04-01

    Full Text Available Identification of pathways involved in the structural transitions of biomolecular systems is often complicated by the transient nature of the conformations visited across energy barriers and the multiplicity of paths accessible in the multidimensional energy landscape. This task becomes even more challenging in exploring molecular systems on the order of megadaltons. Coarse-grained models that lend themselves to analytical solutions appear to be the only possible means of approaching such cases. Motivated by the utility of elastic network models for describing the collective dynamics of biomolecular systems and by the growing theoretical and experimental evidence in support of the intrinsic accessibility of functional substates, we introduce a new method, adaptive anisotropic network model (aANM, for exploring functional transitions. Application to bacterial chaperonin GroEL and comparisons with experimental data, results from action minimization algorithm, and previous simulations support the utility of aANM as a computationally efficient, yet physically plausible, tool for unraveling potential transition pathways sampled by large complexes/assemblies. An important outcome is the assessment of the critical inter-residue interactions formed/broken near the transition state(s, most of which involve conserved residues.

  18. Chaperonin-Inspired pH Protection by Mesoporous Silica SBA-15 on Myoglobin and Lysozyme.

    Science.gov (United States)

    Lynch, Michele M; Liu, Jichuan; Nigra, Michael; Coppens, Marc-Olivier

    2016-09-20

    While enzymes are valuable tools in many fields of biotechnology, they are fragile and must be protected against denaturing conditions such as unfavorable solution pH. Within living organisms, chaperonins help enzymes fold into their native shape and protect them from damage. Inspired by this natural solution, mesoporous silica SBA-15 with different pore diameters is synthesized as a support material for immobilizing and protecting enzymes. In separate experiments, the model enzymes myoglobin and lysozyme are physically adsorbed to SBA-15 and exposed to a range of buffered pH conditions. The immobilized enzymes' biocatalytic activities are quantified and compared to the activities of nonimmobilized enzymes in the same solution conditions. It has been observed that myoglobin immobilized on SBA-15 is protected from acidic denaturation from pH 3.6 to 5.1, exhibiting relative activity of up to 350%. Immobilized lysozyme is protected from unfavorable conditions from pH 6.6 to 7.6, with relative activity of up to 200%. These results indicate that the protective effects conferred to enzymes immobilized by physical adsorption to SBA-15 are driven by the enzymes' electrostatic attraction to the material's surface. The pore diameter of SBA-15 affects the quality of protection given to immobilized enzymes, but the contribution of this effect at different pH values remains unclear.

  19. Hi-C Chromatin Interaction Networks Predict Co-expression in the Mouse Cortex

    Science.gov (United States)

    Hulsman, Marc; Lelieveldt, Boudewijn P. F.; de Ridder, Jeroen; Reinders, Marcel

    2015-01-01

    The three dimensional conformation of the genome in the cell nucleus influences important biological processes such as gene expression regulation. Recent studies have shown a strong correlation between chromatin interactions and gene co-expression. However, predicting gene co-expression from frequent long-range chromatin interactions remains challenging. We address this by characterizing the topology of the cortical chromatin interaction network using scale-aware topological measures. We demonstrate that based on these characterizations it is possible to accurately predict spatial co-expression between genes in the mouse cortex. Consistent with previous findings, we find that the chromatin interaction profile of a gene-pair is a good predictor of their spatial co-expression. However, the accuracy of the prediction can be substantially improved when chromatin interactions are described using scale-aware topological measures of the multi-resolution chromatin interaction network. We conclude that, for co-expression prediction, it is necessary to take into account different levels of chromatin interactions ranging from direct interaction between genes (i.e. small-scale) to chromatin compartment interactions (i.e. large-scale). PMID:25965262

  20. CLIC, a tool for expanding biological pathways based on co-expression across thousands of datasets

    Science.gov (United States)

    Li, Yang; Liu, Jun S.; Mootha, Vamsi K.

    2017-01-01

    In recent years, there has been a huge rise in the number of publicly available transcriptional profiling datasets. These massive compendia comprise billions of measurements and provide a special opportunity to predict the function of unstudied genes based on co-expression to well-studied pathways. Such analyses can be very challenging, however, since biological pathways are modular and may exhibit co-expression only in specific contexts. To overcome these challenges we introduce CLIC, CLustering by Inferred Co-expression. CLIC accepts as input a pathway consisting of two or more genes. It then uses a Bayesian partition model to simultaneously partition the input gene set into coherent co-expressed modules (CEMs), while assigning the posterior probability for each dataset in support of each CEM. CLIC then expands each CEM by scanning the transcriptome for additional co-expressed genes, quantified by an integrated log-likelihood ratio (LLR) score weighted for each dataset. As a byproduct, CLIC automatically learns the conditions (datasets) within which a CEM is operative. We implemented CLIC using a compendium of 1774 mouse microarray datasets (28628 microarrays) or 1887 human microarray datasets (45158 microarrays). CLIC analysis reveals that of 910 canonical biological pathways, 30% consist of strongly co-expressed gene modules for which new members are predicted. For example, CLIC predicts a functional connection between protein C7orf55 (FMC1) and the mitochondrial ATP synthase complex that we have experimentally validated. CLIC is freely available at www.gene-clic.org. We anticipate that CLIC will be valuable both for revealing new components of biological pathways as well as the conditions in which they are active. PMID:28719601

  1. CLIC, a tool for expanding biological pathways based on co-expression across thousands of datasets.

    Directory of Open Access Journals (Sweden)

    Yang Li

    2017-07-01

    Full Text Available In recent years, there has been a huge rise in the number of publicly available transcriptional profiling datasets. These massive compendia comprise billions of measurements and provide a special opportunity to predict the function of unstudied genes based on co-expression to well-studied pathways. Such analyses can be very challenging, however, since biological pathways are modular and may exhibit co-expression only in specific contexts. To overcome these challenges we introduce CLIC, CLustering by Inferred Co-expression. CLIC accepts as input a pathway consisting of two or more genes. It then uses a Bayesian partition model to simultaneously partition the input gene set into coherent co-expressed modules (CEMs, while assigning the posterior probability for each dataset in support of each CEM. CLIC then expands each CEM by scanning the transcriptome for additional co-expressed genes, quantified by an integrated log-likelihood ratio (LLR score weighted for each dataset. As a byproduct, CLIC automatically learns the conditions (datasets within which a CEM is operative. We implemented CLIC using a compendium of 1774 mouse microarray datasets (28628 microarrays or 1887 human microarray datasets (45158 microarrays. CLIC analysis reveals that of 910 canonical biological pathways, 30% consist of strongly co-expressed gene modules for which new members are predicted. For example, CLIC predicts a functional connection between protein C7orf55 (FMC1 and the mitochondrial ATP synthase complex that we have experimentally validated. CLIC is freely available at www.gene-clic.org. We anticipate that CLIC will be valuable both for revealing new components of biological pathways as well as the conditions in which they are active.

  2. The composition, structure and stability of a group II chaperonin are temperature regulated in a hyperthermophilic archaeon.

    Science.gov (United States)

    Kagawa, Hiromi K; Yaoi, Takuro; Brocchieri, Luciano; McMillan, R Andrew; Alton, Thomas; Trent, Jonathan D

    2003-04-01

    The hyperthermoacidophilic archaeon Sulfolobus shibatae contains group II chaperonins, known as rosettasomes, which are two nine-membered rings composed of three different 60 kDa subunits (TF55 alpha, beta and gamma). We sequenced the gene for the gamma subunit and studied the temperature-dependent changes in alpha, beta and gamma expression, their association into rosettasomes and their phylogenetic relationships. Alpha and beta gene expression was increased by heat shock (30 min, 86 degrees C) and decreased by cold shock (30 min, 60 degrees C). Gamma expression was undetectable at heat shock temperatures and low at normal temperatures (75-79 degrees C), but induced by cold shock. Polyacrylamide gel electrophoresis indicated that in vitro alpha and beta subunits form homo-oligomeric rosettasomes, and mixtures of alpha, beta and gamma form hetero-oligomeric rosettasomes. Transmission electron microscopy revealed that beta homo-oligomeric rosettasomes and all hetero-oligomeric rosettasomes associate into filaments. In vivo rosettasomes were hetero-oligomeric with an average subunit ratio of 1alpha:1beta:0.1gamma in cultures grown at 75 degrees C, a ratio of 1alpha:3beta:1gamma in cultures grown at 60 degrees C and a ratio of 2alpha:3beta:0gamma after 86 degrees C heat shock. Using differential scanning calorimetry, we determined denaturation temperatures (Tm) for alpha, beta and gamma subunits of 95.7 degrees C, 96.7 degrees C and 80.5 degrees C, respectively, and observed that rosettasomes containing gamma were relatively less stable than those with alpha and/or beta only. We propose that, in vivo, the rosettasome structure is determined by the relative abundance of subunits and not by a fixed geometry. Furthermore, phylogenetic analyses indicate that archaeal chaperonin subunits underwent multiple duplication events within species (paralogy). The independent evolution of these paralogues raises the possibility that chaperonins have functionally diversified between

  3. Chaperonin-based biolayer interferometry to assess the kinetic stability of metastable, aggregation-prone proteins

    Science.gov (United States)

    Lea, Wendy A.; Naik, Subhashchandra; Chaudhri, Tapan; Machen, Alexandra J.; O’Neil, Pierce T.; McGinn-Straub, Wesley; Tischer, Alexander; Auton, Matthew T.; Burns, Joshua R.; Baldwin, Michael R.; Khar, Karen R.; Karanicolas, John; Fisher, Mark T.

    2017-01-01

    Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy to decrease disease pathologies brought on by protein folding defects or deleterious kinetic transitions. Current methods of examining ligand binding to these marginally stable native states are limited, because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, multi-domain) and metastable proteins (e.g. low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant-pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein immobilized on a BLI biosensor to increasing denaturant concentrations (urea or GnHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remain is detected by increased GroEL binding. Since this kinetic denaturant pulse is brief, the amplitude of the GroEL binding to the immobilized protein depends on the duration of exposure to denaturant, the concentration of denaturant, wash times, and the underlying protein unfolding/refolding kinetics; fixing all other parameters and plotting GroEL binding amplitude versus denaturant pulse concentration results in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein is manifested by a decreased GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be

  4. Uncovering robust patterns of microRNA co-expression across cancers using Bayesian Relevance Networks.

    Directory of Open Access Journals (Sweden)

    Parameswaran Ramachandran

    Full Text Available Co-expression networks have long been used as a tool for investigating the molecular circuitry governing biological systems. However, most algorithms for constructing co-expression networks were developed in the microarray era, before high-throughput sequencing-with its unique statistical properties-became the norm for expression measurement. Here we develop Bayesian Relevance Networks, an algorithm that uses Bayesian reasoning about expression levels to account for the differing levels of uncertainty in expression measurements between highly- and lowly-expressed entities, and between samples with different sequencing depths. It combines data from groups of samples (e.g., replicates to estimate group expression levels and confidence ranges. It then computes uncertainty-moderated estimates of cross-group correlations between entities, and uses permutation testing to assess their statistical significance. Using large scale miRNA data from The Cancer Genome Atlas, we show that our Bayesian update of the classical Relevance Networks algorithm provides improved reproducibility in co-expression estimates and lower false discovery rates in the resulting co-expression networks. Software is available at www.perkinslab.ca.

  5. The Detection of Metabolite-Mediated Gene Module Co-Expression Using Multivariate Linear Models.

    Directory of Open Access Journals (Sweden)

    Trishanta Padayachee

    Full Text Available Investigating whether metabolites regulate the co-expression of a predefined gene module is one of the relevant questions posed in the integrative analysis of metabolomic and transcriptomic data. This article concerns the integrative analysis of the two high-dimensional datasets by means of multivariate models and statistical tests for the dependence between metabolites and the co-expression of a gene module. The general linear model (GLM for correlated data that we propose models the dependence between adjusted gene expression values through a block-diagonal variance-covariance structure formed by metabolic-subset specific general variance-covariance blocks. Performance of statistical tests for the inference of conditional co-expression are evaluated through a simulation study. The proposed methodology is applied to the gene expression data of the previously characterized lipid-leukocyte module. Our results show that the GLM approach improves on a previous approach by being less prone to the detection of spurious conditional co-expression.

  6. Co-expression Network Approach Reveals Functional Similarities among Diseases Affecting Human Skeletal Muscle

    OpenAIRE

    Kavitha Mukund; Shankar Subramaniam

    2017-01-01

    Diseases affecting skeletal muscle exhibit considerable heterogeneity in intensity, etiology, phenotypic manifestation and gene expression. Systems biology approaches using network theory, allows for a holistic understanding of functional similarities amongst diseases. Here we propose a co-expression based, network theoretic approach to extract functional similarities from 20 heterogeneous diseases comprising of dystrophinopathies, inflammatory myopathies, neuromuscular, and muscle metabolic ...

  7. Genome-wide tissue-specific gene expression, co-expression and regulation of co-expressed genes in adult nematode Ascaris suum.

    Science.gov (United States)

    Rosa, Bruce A; Jasmer, Douglas P; Mitreva, Makedonka

    2014-02-01

    Caenorhabditis elegans has traditionally been used as a model for studying nematode biology, but its small size limits the ability for researchers to perform some experiments such as high-throughput tissue-specific gene expression studies. However, the dissection of individual tissues is possible in the parasitic nematode Ascaris suum due to its relatively large size. Here, we take advantage of the recent genome sequencing of Ascaris suum and the ability to physically dissect its separate tissues to produce a wide-scale tissue-specific nematode RNA-seq datasets, including data on three non-reproductive tissues (head, pharynx, and intestine) in both male and female worms, as well as four reproductive tissues (testis, seminal vesicle, ovary, and uterus). We obtained fundamental information about the biology of diverse cell types and potential interactions among tissues within this multicellular organism. Overexpression and functional enrichment analyses identified many putative biological functions enriched in each tissue studied, including functions which have not been previously studied in detail in nematodes. Putative tissue-specific transcriptional factors and corresponding binding motifs that regulate expression in each tissue were identified, including the intestine-enriched ELT-2 motif/transcription factor previously described in nematode intestines. Constitutively expressed and novel genes were also characterized, with the largest number of novel genes found to be overexpressed in the testis. Finally, a putative acetylcholine-mediated transcriptional network connecting biological activity in the head to the male reproductive system is described using co-expression networks, along with a similar ecdysone-mediated system in the female. The expression profiles, co-expression networks and co-expression regulation of the 10 tissues studied and the tissue-specific analysis presented here are a valuable resource for studying tissue-specific biological functions in

  8. Genome-wide tissue-specific gene expression, co-expression and regulation of co-expressed genes in adult nematode Ascaris suum.

    Directory of Open Access Journals (Sweden)

    Bruce A Rosa

    2014-02-01

    Full Text Available Caenorhabditis elegans has traditionally been used as a model for studying nematode biology, but its small size limits the ability for researchers to perform some experiments such as high-throughput tissue-specific gene expression studies. However, the dissection of individual tissues is possible in the parasitic nematode Ascaris suum due to its relatively large size. Here, we take advantage of the recent genome sequencing of Ascaris suum and the ability to physically dissect its separate tissues to produce a wide-scale tissue-specific nematode RNA-seq datasets, including data on three non-reproductive tissues (head, pharynx, and intestine in both male and female worms, as well as four reproductive tissues (testis, seminal vesicle, ovary, and uterus. We obtained fundamental information about the biology of diverse cell types and potential interactions among tissues within this multicellular organism.Overexpression and functional enrichment analyses identified many putative biological functions enriched in each tissue studied, including functions which have not been previously studied in detail in nematodes. Putative tissue-specific transcriptional factors and corresponding binding motifs that regulate expression in each tissue were identified, including the intestine-enriched ELT-2 motif/transcription factor previously described in nematode intestines. Constitutively expressed and novel genes were also characterized, with the largest number of novel genes found to be overexpressed in the testis. Finally, a putative acetylcholine-mediated transcriptional network connecting biological activity in the head to the male reproductive system is described using co-expression networks, along with a similar ecdysone-mediated system in the female.The expression profiles, co-expression networks and co-expression regulation of the 10 tissues studied and the tissue-specific analysis presented here are a valuable resource for studying tissue

  9. Constructing gene co-expression networks and predicting functions of unknown genes by random matrix theory

    Science.gov (United States)

    Luo, Feng; Yang, Yunfeng; Zhong, Jianxin; Gao, Haichun; Khan, Latifur; Thompson, Dorothea K; Zhou, Jizhong

    2007-01-01

    Background Large-scale sequencing of entire genomes has ushered in a new age in biology. One of the next grand challenges is to dissect the cellular networks consisting of many individual functional modules. Defining co-expression networks without ambiguity based on genome-wide microarray data is difficult and current methods are not robust and consistent with different data sets. This is particularly problematic for little understood organisms since not much existing biological knowledge can be exploited for determining the threshold to differentiate true correlation from random noise. Random matrix theory (RMT), which has been widely and successfully used in physics, is a powerful approach to distinguish system-specific, non-random properties embedded in complex systems from random noise. Here, we have hypothesized that the universal predictions of RMT are also applicable to biological systems and the correlation threshold can be determined by characterizing the correlation matrix of microarray profiles using random matrix theory. Results Application of random matrix theory to microarray data of S. oneidensis, E. coli, yeast, A. thaliana, Drosophila, mouse and human indicates that there is a sharp transition of nearest neighbour spacing distribution (NNSD) of correlation matrix after gradually removing certain elements insider the matrix. Testing on an in silico modular model has demonstrated that this transition can be used to determine the correlation threshold for revealing modular co-expression networks. The co-expression network derived from yeast cell cycling microarray data is supported by gene annotation. The topological properties of the resulting co-expression network agree well with the general properties of biological networks. Computational evaluations have showed that RMT approach is sensitive and robust. Furthermore, evaluation on sampled expression data of an in silico modular gene system has showed that under-sampled expressions do not affect the

  10. Constructing gene co-expression networks and predicting functions of unknown genes by random matrix theory.

    Science.gov (United States)

    Luo, Feng; Yang, Yunfeng; Zhong, Jianxin; Gao, Haichun; Khan, Latifur; Thompson, Dorothea K; Zhou, Jizhong

    2007-08-14

    Large-scale sequencing of entire genomes has ushered in a new age in biology. One of the next grand challenges is to dissect the cellular networks consisting of many individual functional modules. Defining co-expression networks without ambiguity based on genome-wide microarray data is difficult and current methods are not robust and consistent with different data sets. This is particularly problematic for little understood organisms since not much existing biological knowledge can be exploited for determining the threshold to differentiate true correlation from random noise. Random matrix theory (RMT), which has been widely and successfully used in physics, is a powerful approach to distinguish system-specific, non-random properties embedded in complex systems from random noise. Here, we have hypothesized that the universal predictions of RMT are also applicable to biological systems and the correlation threshold can be determined by characterizing the correlation matrix of microarray profiles using random matrix theory. Application of random matrix theory to microarray data of S. oneidensis, E. coli, yeast, A. thaliana, Drosophila, mouse and human indicates that there is a sharp transition of nearest neighbour spacing distribution (NNSD) of correlation matrix after gradually removing certain elements insider the matrix. Testing on an in silico modular model has demonstrated that this transition can be used to determine the correlation threshold for revealing modular co-expression networks. The co-expression network derived from yeast cell cycling microarray data is supported by gene annotation. The topological properties of the resulting co-expression network agree well with the general properties of biological networks. Computational evaluations have showed that RMT approach is sensitive and robust. Furthermore, evaluation on sampled expression data of an in silico modular gene system has showed that under-sampled expressions do not affect the recovery of gene

  11. Large-scale co-expression approach to dissect secondary cell wall formation across plant species

    Directory of Open Access Journals (Sweden)

    Colin eRuprecht

    2011-07-01

    Full Text Available Plant cell walls are complex composites largely consisting of carbohydrate-based polymers, and are generally divided into primary and secondary walls based on content and characteristics. Cellulose microfibrils constitute a major component of both primary and secondary cell walls and are synthesized at the plasma membrane by cellulose synthase (CESA complexes. Several studies in Arabidopsis have demonstrated the power of co-expression analyses to identify new genes associated with secondary wall cellulose biosynthesis. However, across-species comparative co-expression analyses remain largely unexplored. Here, we compared co-expressed gene vicinity networks of primary and secondary wall CESAs in Arabidopsis, barley, rice, poplar, soybean, Medicago and wheat, and identified gene families that are consistently co-regulated with cellulose biosynthesis. In addition to the expected polysaccharide acting enzymes, we also found many gene families associated with cytoskeleton, signaling, transcriptional regulation, oxidation and protein degradation. Based on these analyses, we selected and biochemically analyzed T-DNA insertion lines corresponding to approximately twenty genes from gene families that re-occur in the co-expressed gene vicinity networks of secondary wall CESAs across the seven species. We developed a statistical pipeline using principal component analysis (PCA and optimal clustering based on silhouette width to analyze sugar profiles. One of the mutants, corresponding to a pinoresinol reductase gene, displayed disturbed xylem morphology and held lower levels of lignin molecules. We propose that this type of large-scale co-expression approach, coupled with statistical analysis of the cell wall contents, will be useful to facilitate rapid knowledge transfer across plant species.

  12. Comparison of co-expression measures: mutual information, correlation, and model based indices

    Science.gov (United States)

    2012-01-01

    Background Co-expression measures are often used to define networks among genes. Mutual information (MI) is often used as a generalized correlation measure. It is not clear how much MI adds beyond standard (robust) correlation measures or regression model based association measures. Further, it is important to assess what transformations of these and other co-expression measures lead to biologically meaningful modules (clusters of genes). Results We provide a comprehensive comparison between mutual information and several correlation measures in 8 empirical data sets and in simulations. We also study different approaches for transforming an adjacency matrix, e.g. using the topological overlap measure. Overall, we confirm close relationships between MI and correlation in all data sets which reflects the fact that most gene pairs satisfy linear or monotonic relationships. We discuss rare situations when the two measures disagree. We also compare correlation and MI based approaches when it comes to defining co-expression network modules. We show that a robust measure of correlation (the biweight midcorrelation transformed via the topological overlap transformation) leads to modules that are superior to MI based modules and maximal information coefficient (MIC) based modules in terms of gene ontology enrichment. We present a function that relates correlation to mutual information which can be used to approximate the mutual information from the corresponding correlation coefficient. We propose the use of polynomial or spline regression models as an alternative to MI for capturing non-linear relationships between quantitative variables. Conclusion The biweight midcorrelation outperforms MI in terms of elucidating gene pairwise relationships. Coupled with the topological overlap matrix transformation, it often leads to more significantly enriched co-expression modules. Spline and polynomial networks form attractive alternatives to MI in case of non-linear relationships

  13. Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum

    Science.gov (United States)

    Wu, Yong-Qing; Jiang, Pei-Hong; Fan, Chang-Sheng; Wang, Jian-Gang; Shang, Liang; Huang, Wei-Da

    2003-01-01

    AIM: To study the effect of co-expression of ppsA, pckA, aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strain for L-phenylalanine. METHODS: ppsA and pckA genes were amplified from genomic DNA of E. coli by polymerase chain reaction, and then introduced into shuttle vectors between E coli and Brevibacterium flavum to generate constructs pJN2 and pJN5. pJN2 was generated by inserting ppsA and pckA genes into vector pCZ; whereas pJN5 was obtained by introducing ppsA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes. The recombinant plasmids were then introduced into B. flavum by electroporation and the transformants were used for L-phenylalanine fermentation. RESULTS: Compared with the original B. flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably. pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold. Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly. CONCLUSION: Co-expression of ppsA and pckA genes in B. flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pckA, aroG, pheA and tyrB of E. coli in B. flavum was a feasible approach to construct a strain for phenylalanine production. PMID:12532463

  14. Chaperonin 20 might be an iron chaperone for superoxide dismutase in activating iron superoxide dismutase (FeSOD).

    Science.gov (United States)

    Kuo, Wen-Yu; Huang, Chien-Hsun; Jinn, Tsung-Luo

    2013-02-01

    Activation of Cu/Zn superoxide dismutases (CuZnSODs) is aided by Cu incorporation and disulfide isomerization by Cu chaperone of SOD (CCS). As well, an Fe-S cluster scaffold protein, ISU, might alter the incorporation of Fe or Mn into yeast MnSOD (ySOD2), thus leading to active or inactive ySOD2. However, metallochaperones involved in the activation of FeSODs are unknown. Recently, we found that a chloroplastic chaperonin cofactor, CPN20, could mediate FeSOD activity. To investigate whether Fe incorporation in FeSOD is affected by CPN20, we used inductively coupled plasma mass spectrometry to analyze the ability of CPN20 to bind Fe. CPN20 could bind Fe, and the Fe binding to FeSOD was increased with CPN20 incubation. Thus, CPN20 might be an Fe chaperone for FeSOD activation, a role independent of its well-known co-chaperonin activity.

  15. Disease-Associated Mutations in the HSPD1 Gene Encoding the Large Subunit of the Mitochondrial HSP60/HSP10 Chaperonin Complex

    DEFF Research Database (Denmark)

    Bross, Peter; Fernandez-Guerra, Paula

    2016-01-01

    Heat shock protein 60 (HSP60) forms together with heat shock protein 10 (HSP10) double-barrel chaperonin complexes that are essential for folding to the native state of proteins in the mitochondrial matrix space. Two extremely rare monogenic disorders have been described that are caused by missen...

  16. Effects of a Mutation in the HSPE1 Gene Encoding the Mitochondrial Co-chaperonin HSP10 and Its Potential Association with a Neurological and Developmental Disorder

    DEFF Research Database (Denmark)

    Bie, Anne S; Fernandez-Guerra, Paula; Birkler, Rune I D

    2016-01-01

    We here report molecular investigations of a missense mutation in the HSPE1 gene encoding the HSP10 subunit of the HSP60/ HSP10 chaperonin complex that assists protein folding in the mitochondrial matrix. The mutation was identified in an infant who came to clinical attention due to infantile spa...

  17. Chaperonin GroEL/GroES Over-Expression Promotes Aminoglycoside Resistance and Reduces Drug Susceptibilities in Escherichia coli Following Exposure to Sublethal Aminoglycoside Doses

    DEFF Research Database (Denmark)

    Goltermann, Lise; Sarusie, Menachem V; Bentin, Thomas

    2016-01-01

    Antibiotic resistance is an increasing challenge to modern healthcare. Aminoglycoside antibiotics cause translation corruption and protein misfolding and aggregation in Escherichia coli. We previously showed that chaperonin GroEL/GroES depletion and over-expression sensitize and promote short...

  18. Bardet-Biedl Syndrome as a Chaperonopathy: Dissecting the Major Role of Chaperonin-Like BBS Proteins (BBS6-BBS10-BBS12

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    María Álvarez-Satta

    2017-07-01

    Full Text Available Bardet-Biedl syndrome (BBS is a rare genetic disorder that belongs to the group of ciliopathies, defined as diseases caused by defects in cilia structure and/or function. The six diagnostic features considered for this syndrome include retinal dystrophy, obesity, polydactyly, cognitive impairment and renal and urogenital anomalies. Furthermore, three of the 21 genes currently known to be involved in BBS encode chaperonin-like proteins (MKKS/BBS6, BBS10, and BBS12, so BBS can be also considered a member of the growing group of chaperonopathies. Remarkably, up to 50% of clinically-diagnosed BBS families can harbor disease-causing variants in these three genes, which highlights the importance of chaperone defects as pathogenic factors even for genetically heterogeneous syndromes such as BBS. In addition, it is interesting to note that BBS families with deleterious variants in MKKS/BBS6, BBS10 or BBS12 genes generally display more severe phenotypes than families with changes in other BBS genes. The chaperonin-like BBS proteins have structural homology to the CCT family of group II chaperonins, although they are believed to conserve neither the ATP-dependent folding activity of canonical CCT chaperonins nor the ability to form CCT-like oligomeric complexes. Thus, they play an important role in the initial steps of assembly of the BBSome, which is a multiprotein complex essential for mediating the ciliary trafficking activity. In this review, we present a comprehensive review of those genetic, functional and evolutionary aspects concerning chaperonin-like BBS proteins, trying to provide a new perspective that expands the classical conception of BBS only from a ciliary point of view.

  19. Eigengene networks for studying the relationships between co-expression modules

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    Horvath Steve

    2007-11-01

    Full Text Available Abstract Background There is evidence that genes and their protein products are organized into functional modules according to cellular processes and pathways. Gene co-expression networks have been used to describe the relationships between gene transcripts. Ample literature exists on how to detect biologically meaningful modules in networks but there is a need for methods that allow one to study the relationships between modules. Results We show that network methods can also be used to describe the relationships between co-expression modules and present the following methodology. First, we describe several methods for detecting modules that are shared by two or more networks (referred to as consensus modules. We represent the gene expression profiles of each module by an eigengene. Second, we propose a method for constructing an eigengene network, where the edges are undirected but maintain information on the sign of the co-expression information. Third, we propose methods for differential eigengene network analysis that allow one to assess the preservation of network properties across different data sets. We illustrate the value of eigengene networks in studying the relationships between consensus modules in human and chimpanzee brains; the relationships between consensus modules in brain, muscle, liver, and adipose mouse tissues; and the relationships between male-female mouse consensus modules and clinical traits. In some applications, we find that module eigengenes can be organized into higher level clusters which we refer to as meta-modules. Conclusion Eigengene networks can be effective and biologically meaningful tools for studying the relationships between modules of a gene co-expression network. The proposed methods may reveal a higher order organization of the transcriptome. R software tutorials, the data, and supplementary material can be found at the following webpage: http://www.genetics.ucla.edu/labs/horvath/CoexpressionNetwork/EigengeneNetwork.

  20. Module discovery by exhaustive search for densely connected, co-expressed regions in biomolecular interaction networks.

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    Recep Colak

    2010-10-01

    Full Text Available Computational prediction of functionally related groups of genes (functional modules from large-scale data is an important issue in computational biology. Gene expression experiments and interaction networks are well studied large-scale data sources, available for many not yet exhaustively annotated organisms. It has been well established, when analyzing these two data sources jointly, modules are often reflected by highly interconnected (dense regions in the interaction networks whose participating genes are co-expressed. However, the tractability of the problem had remained unclear and methods by which to exhaustively search for such constellations had not been presented.We provide an algorithmic framework, referred to as Densely Connected Biclustering (DECOB, by which the aforementioned search problem becomes tractable. To benchmark the predictive power inherent to the approach, we computed all co-expressed, dense regions in physical protein and genetic interaction networks from human and yeast. An automatized filtering procedure reduces our output which results in smaller collections of modules, comparable to state-of-the-art approaches. Our results performed favorably in a fair benchmarking competition which adheres to standard criteria. We demonstrate the usefulness of an exhaustive module search, by using the unreduced output to more quickly perform GO term related function prediction tasks. We point out the advantages of our exhaustive output by predicting functional relationships using two examples.We demonstrate that the computation of all densely connected and co-expressed regions in interaction networks is an approach to module discovery of considerable value. Beyond confirming the well settled hypothesis that such co-expressed, densely connected interaction network regions reflect functional modules, we open up novel computational ways to comprehensively analyze the modular organization of an organism based on prevalent and largely

  1. An extensive (co-expression analysis tool for the cytochrome P450 superfamily in Arabidopsis thaliana

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    Provart Nicholas J

    2008-04-01

    Full Text Available Abstract Background Sequencing of the first plant genomes has revealed that cytochromes P450 have evolved to become the largest family of enzymes in secondary metabolism. The proportion of P450 enzymes with characterized biochemical function(s is however very small. If P450 diversification mirrors evolution of chemical diversity, this points to an unexpectedly poor understanding of plant metabolism. We assumed that extensive analysis of gene expression might guide towards the function of P450 enzymes, and highlight overlooked aspects of plant metabolism. Results We have created a comprehensive database, 'CYPedia', describing P450 gene expression in four data sets: organs and tissues, stress response, hormone response, and mutants of Arabidopsis thaliana, based on public Affymetrix ATH1 microarray expression data. P450 expression was then combined with the expression of 4,130 re-annotated genes, predicted to act in plant metabolism, for co-expression analyses. Based on the annotation of co-expressed genes from diverse pathway annotation databases, co-expressed pathways were identified. Predictions were validated for most P450s with known functions. As examples, co-expression results for P450s related to plastidial functions/photosynthesis, and to phenylpropanoid, triterpenoid and jasmonate metabolism are highlighted here. Conclusion The large scale hypothesis generation tools presented here provide leads to new pathways, unexpected functions, and regulatory networks for many P450s in plant metabolism. These can now be exploited by the community to validate the proposed functions experimentally using reverse genetics, biochemistry, and metabolic profiling.

  2. Identification of Drosophila mitotic genes by combining co-expression analysis and RNA interference.

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    Maria Patrizia Somma

    2008-07-01

    Full Text Available RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression-based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression-based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins.

  3. Genome-wide patterns of promoter sharing and co-expression in bovine skeletal muscle

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    Dalrymple Brian P

    2011-01-01

    Full Text Available Abstract Background Gene regulation by transcription factors (TF is species, tissue and time specific. To better understand how the genetic code controls gene expression in bovine muscle we associated gene expression data from developing Longissimus thoracis et lumborum skeletal muscle with bovine promoter sequence information. Results We created a highly conserved genome-wide promoter landscape comprising 87,408 interactions relating 333 TFs with their 9,242 predicted target genes (TGs. We discovered that the complete set of predicted TGs share an average of 2.75 predicted TF binding sites (TFBSs and that the average co-expression between a TF and its predicted TGs is higher than the average co-expression between the same TF and all genes. Conversely, pairs of TFs sharing predicted TGs showed a co-expression correlation higher that pairs of TFs not sharing TGs. Finally, we exploited the co-occurrence of predicted TFBS in the context of muscle-derived functionally-coherent modules including cell cycle, mitochondria, immune system, fat metabolism, muscle/glycolysis, and ribosome. Our findings enabled us to reverse engineer a regulatory network of core processes, and correctly identified the involvement of E2F1, GATA2 and NFKB1 in the regulation of cell cycle, fat, and muscle/glycolysis, respectively. Conclusion The pivotal implication of our research is two-fold: (1 there exists a robust genome-wide expression signal between TFs and their predicted TGs in cattle muscle consistent with the extent of promoter sharing; and (2 this signal can be exploited to recover the cellular mechanisms underpinning transcription regulation of muscle structure and development in bovine. Our study represents the first genome-wide report linking tissue specific co-expression to co-regulation in a non-model vertebrate.

  4. FastGCN: a GPU accelerated tool for fast gene co-expression networks.

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    Meimei Liang

    Full Text Available Gene co-expression networks comprise one type of valuable biological networks. Many methods and tools have been published to construct gene co-expression networks; however, most of these tools and methods are inconvenient and time consuming for large datasets. We have developed a user-friendly, accelerated and optimized tool for constructing gene co-expression networks that can fully harness the parallel nature of GPU (Graphic Processing Unit architectures. Genetic entropies were exploited to filter out genes with no or small expression changes in the raw data preprocessing step. Pearson correlation coefficients were then calculated. After that, we normalized these coefficients and employed the False Discovery Rate to control the multiple tests. At last, modules identification was conducted to construct the co-expression networks. All of these calculations were implemented on a GPU. We also compressed the coefficient matrix to save space. We compared the performance of the GPU implementation with those of multi-core CPU implementations with 16 CPU threads, single-thread C/C++ implementation and single-thread R implementation. Our results show that GPU implementation largely outperforms single-thread C/C++ implementation and single-thread R implementation, and GPU implementation outperforms multi-core CPU implementation when the number of genes increases. With the test dataset containing 16,000 genes and 590 individuals, we can achieve greater than 63 times the speed using a GPU implementation compared with a single-thread R implementation when 50 percent of genes were filtered out and about 80 times the speed when no genes were filtered out.

  5. FastGCN: a GPU accelerated tool for fast gene co-expression networks.

    Science.gov (United States)

    Liang, Meimei; Zhang, Futao; Jin, Gulei; Zhu, Jun

    2015-01-01

    Gene co-expression networks comprise one type of valuable biological networks. Many methods and tools have been published to construct gene co-expression networks; however, most of these tools and methods are inconvenient and time consuming for large datasets. We have developed a user-friendly, accelerated and optimized tool for constructing gene co-expression networks that can fully harness the parallel nature of GPU (Graphic Processing Unit) architectures. Genetic entropies were exploited to filter out genes with no or small expression changes in the raw data preprocessing step. Pearson correlation coefficients were then calculated. After that, we normalized these coefficients and employed the False Discovery Rate to control the multiple tests. At last, modules identification was conducted to construct the co-expression networks. All of these calculations were implemented on a GPU. We also compressed the coefficient matrix to save space. We compared the performance of the GPU implementation with those of multi-core CPU implementations with 16 CPU threads, single-thread C/C++ implementation and single-thread R implementation. Our results show that GPU implementation largely outperforms single-thread C/C++ implementation and single-thread R implementation, and GPU implementation outperforms multi-core CPU implementation when the number of genes increases. With the test dataset containing 16,000 genes and 590 individuals, we can achieve greater than 63 times the speed using a GPU implementation compared with a single-thread R implementation when 50 percent of genes were filtered out and about 80 times the speed when no genes were filtered out.

  6. Co-expression of activin receptor-interacting protein 1 and 2 in mouse nerve cells.

    Science.gov (United States)

    Qi, Yan; Ge, Jing-Yan; Wang, Yi-Nan; Liu, Hai-Yan; Li, Yun-Man; Liu, Zhong-Hui; Cui, Xue-Ling

    2013-05-10

    Activin is a neurotrophic and neuroprotective factor in the central nervous system. Activin receptor-interacting protein 1 and 2 (ARIP1 and ARIP2) are identified as activin signal proteins in mouse brain. However, whether ARIP1 and ARIP2 are co-expressed in nerve cells and the differences of their biological activities are not well characterized. In the present study, we found that ARIP1 and ARIP2 mRNA expressions were detectable in mouse brain and their proteins were co-localized at the hypothalamus of cerebrum and granular layers in cerebellum, especially in Purkinje cells. Furthermore, ARIP1 and ARIP2 were co-expressed in mouse Neuro-2a cells, which is similar to the co-localization of ARIP1 and ARIP2 in hypothalamus neurons and Purkinje cells. Overexpression of ARIP1 in Neuro-2a cells inhibited activin signal transduction induced by activin A and Smad3, and activin A-induced voltage-gated Na(+) current (INa), while ARIP2 was only a negative regulator of signal transduction induced by activin A and did not alter activin A-induced INa. Taken together, these data demonstrate that ARIP1 and ARIP2 are co-expressed in some nerve cells and their biological activities are distinct. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  7. Massive-Scale Gene Co-Expression Network Construction and Robustness Testing Using Random Matrix Theory

    Science.gov (United States)

    Isaacson, Sven; Luo, Feng; Feltus, Frank A.; Smith, Melissa C.

    2013-01-01

    The study of gene relationships and their effect on biological function and phenotype is a focal point in systems biology. Gene co-expression networks built using microarray expression profiles are one technique for discovering and interpreting gene relationships. A knowledge-independent thresholding technique, such as Random Matrix Theory (RMT), is useful for identifying meaningful relationships. Highly connected genes in the thresholded network are then grouped into modules that provide insight into their collective functionality. While it has been shown that co-expression networks are biologically relevant, it has not been determined to what extent any given network is functionally robust given perturbations in the input sample set. For such a test, hundreds of networks are needed and hence a tool to rapidly construct these networks. To examine functional robustness of networks with varying input, we enhanced an existing RMT implementation for improved scalability and tested functional robustness of human (Homo sapiens), rice (Oryza sativa) and budding yeast (Saccharomyces cerevisiae). We demonstrate dramatic decrease in network construction time and computational requirements and show that despite some variation in global properties between networks, functional similarity remains high. Moreover, the biological function captured by co-expression networks thresholded by RMT is highly robust. PMID:23409071

  8. Discovering missing reactions of metabolic networks by using gene co-expression data

    Science.gov (United States)

    Hosseini, Zhaleh; Marashi, Sayed-Amir

    2017-02-01

    Flux coupling analysis is a computational method which is able to explain co-expression of metabolic genes by analyzing the topological structure of a metabolic network. It has been suggested that if genes in two seemingly fully-coupled reactions are not highly co-expressed, then these two reactions are not fully coupled in reality, and hence, there is a gap or missing reaction in the network. Here, we present GAUGE as a novel approach for gap filling of metabolic networks, which is a two-step algorithm based on a mixed integer linear programming formulation. In GAUGE, the discrepancies between experimental co-expression data and predicted flux coupling relations is minimized by adding a minimum number of reactions to the network. We show that GAUGE is able to predict missing reactions of E. coli metabolism that are not detectable by other popular gap filling approaches. We propose that our algorithm may be used as a complementary strategy for the gap filling problem of metabolic networks. Since GAUGE relies only on gene expression data, it can be potentially useful for exploring missing reactions in the metabolism of non-model organisms, which are often poorly characterized, cannot grow in the laboratory, and lack genetic tools for generating knockouts.

  9. Constructing a comprehensive gene co-expression based interactome in Bos taurus.

    Science.gov (United States)

    Chen, Yan; Liu, Yining; Du, Min; Zhang, Wengang; Xu, Ling; Gao, Xue; Zhang, Lupei; Gao, Huijiang; Xu, Lingyang; Li, Junya; Zhao, Min

    2017-01-01

    Integrating genomic information into cattle breeding is an important approach to exploring genotype-phenotype relationships for complex traits related to diary and meat production. To assist with genomic-based selection, a reference map of interactome is needed to fully understand and identify the functional relevant genes. To this end, we constructed a co-expression analysis of 92 tissues and this represents the systematic exploration of gene-gene relationship in Bos taurus. By using robust WGCNA (Weighted Gene Correlation Network Analysis), we described the gene co-expression network of 5,000 protein-coding genes with majority variations in expression across 92 tissues. Further module identifications found 55 highly organized functional clusters representing diverse cellular activities. To demonstrate the re-use of our interaction for functional genomics analysis, we extracted a sub-network associated with DNA binding genes in Bos taurus. The subnetwork was enriched within regulation of transcription from RNA polymerase II promoter representing central cellular functions. In addition, we identified 28 novel linker genes associated with more than 100 DNA binding genes. Our WGCNA-based co-expression network reconstruction will be a valuable resource for exploring the molecular mechanisms of incompletely characterized proteins and for elucidating larger-scale patterns of functional modulization in the Bos taurus genome.

  10. Co-expressed Pathways DataBase for Tomato: a database to predict pathways relevant to a query gene.

    Science.gov (United States)

    Narise, Takafumi; Sakurai, Nozomu; Obayashi, Takeshi; Ohta, Hiroyuki; Shibata, Daisuke

    2017-06-05

    Gene co-expression, the similarity of gene expression profiles under various experimental conditions, has been used as an indicator of functional relationships between genes, and many co-expression databases have been developed for predicting gene functions. These databases usually provide users with a co-expression network and a list of strongly co-expressed genes for a query gene. Several of these databases also provide functional information on a set of strongly co-expressed genes (i.e., provide biological processes and pathways that are enriched in these strongly co-expressed genes), which is generally analyzed via over-representation analysis (ORA). A limitation of this approach may be that users can predict gene functions only based on the strongly co-expressed genes. In this study, we developed a new co-expression database that enables users to predict the function of tomato genes from the results of functional enrichment analyses of co-expressed genes while considering the genes that are not strongly co-expressed. To achieve this, we used the ORA approach with several thresholds to select co-expressed genes, and performed gene set enrichment analysis (GSEA) applied to a ranked list of genes ordered by the co-expression degree. We found that internal correlation in pathways affected the significance levels of the enrichment analyses. Therefore, we introduced a new measure for evaluating the relationship between the gene and pathway, termed the percentile (p)-score, which enables users to predict functionally relevant pathways without being affected by the internal correlation in pathways. In addition, we evaluated our approaches using receiver operating characteristic curves, which concluded that the p-score could improve the performance of the ORA. We developed a new database, named Co-expressed Pathways DataBase for Tomato, which is available at http://cox-path-db.kazusa.or.jp/tomato . The database allows users to predict pathways that are relevant to a

  11. Differentially correlated genes in co-expression networks control phenotype transitions [version 1; referees: 1 approved, 2 approved with reservations

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    Lina D. Thomas

    2016-11-01

    Full Text Available Background: Co-expression networks are a tool widely used for analysis of “Big Data” in biology that can range from transcriptomes to proteomes, metabolomes and more recently even microbiomes. Several methods were proposed to answer biological questions interrogating these networks. Differential co-expression analysis is a recent approach that measures how gene interactions change when a biological system transitions from one state to another. Although the importance of differentially co-expressed genes to identify dysregulated pathways has been noted, their role in gene regulation is not well studied. Herein we investigated differentially co-expressed genes in a relatively simple mono-causal process (B lymphocyte deficiency and in a complex multi-causal system (cervical cancer. Methods: Co-expression networks of B cell deficiency (Control and BcKO were reconstructed using Pearson correlation coefficient for two mus musculus datasets: B10.A strain (12 normal, 12 BcKO and BALB/c strain (10 normal, 10 BcKO. Co-expression networks of cervical cancer (normal and cancer were reconstructed using local partial correlation method for five datasets (total of 64 normal, 148 cancer. Differentially correlated pairs were identified along with the location of their genes in BcKO and in cancer networks. Minimum Shortest Path and Bi-partite Betweenness Centrality where statistically evaluated for differentially co-expressed genes in corresponding networks.    Results: We show that in B cell deficiency the differentially co-expressed genes are highly enriched with immunoglobulin genes (causal genes. In cancer we found that differentially co-expressed genes act as “bottlenecks” rather than causal drivers with most flows that come from the key driver genes to the peripheral genes passing through differentially co-expressed genes. Using in vitro knockdown experiments for two out of 14 differentially co-expressed genes found in cervical cancer (FGFR2 and

  12. Annotation of gene function in citrus using gene expression information and co-expression networks.

    Science.gov (United States)

    Wong, Darren C J; Sweetman, Crystal; Ford, Christopher M

    2014-07-15

    The genus Citrus encompasses major cultivated plants such as sweet orange, mandarin, lemon and grapefruit, among the world's most economically important fruit crops. With increasing volumes of transcriptomics data available for these species, Gene Co-expression Network (GCN) analysis is a viable option for predicting gene function at a genome-wide scale. GCN analysis is based on a "guilt-by-association" principle whereby genes encoding proteins involved in similar and/or related biological processes may exhibit similar expression patterns across diverse sets of experimental conditions. While bioinformatics resources such as GCN analysis are widely available for efficient gene function prediction in model plant species including Arabidopsis, soybean and rice, in citrus these tools are not yet developed. We have constructed a comprehensive GCN for citrus inferred from 297 publicly available Affymetrix Genechip Citrus Genome microarray datasets, providing gene co-expression relationships at a genome-wide scale (33,000 transcripts). The comprehensive citrus GCN consists of a global GCN (condition-independent) and four condition-dependent GCNs that survey the sweet orange species only, all citrus fruit tissues, all citrus leaf tissues, or stress-exposed plants. All of these GCNs are clustered using genome-wide, gene-centric (guide) and graph clustering algorithms for flexibility of gene function prediction. For each putative cluster, gene ontology (GO) enrichment and gene expression specificity analyses were performed to enhance gene function, expression and regulation pattern prediction. The guide-gene approach was used to infer novel roles of genes involved in disease susceptibility and vitamin C metabolism, and graph-clustering approaches were used to investigate isoprenoid/phenylpropanoid metabolism in citrus peel, and citric acid catabolism via the GABA shunt in citrus fruit. Integration of citrus gene co-expression networks, functional enrichment analysis and gene

  13. Annotation of gene function in citrus using gene expression information and co-expression networks

    Science.gov (United States)

    2014-01-01

    Background The genus Citrus encompasses major cultivated plants such as sweet orange, mandarin, lemon and grapefruit, among the world’s most economically important fruit crops. With increasing volumes of transcriptomics data available for these species, Gene Co-expression Network (GCN) analysis is a viable option for predicting gene function at a genome-wide scale. GCN analysis is based on a “guilt-by-association” principle whereby genes encoding proteins involved in similar and/or related biological processes may exhibit similar expression patterns across diverse sets of experimental conditions. While bioinformatics resources such as GCN analysis are widely available for efficient gene function prediction in model plant species including Arabidopsis, soybean and rice, in citrus these tools are not yet developed. Results We have constructed a comprehensive GCN for citrus inferred from 297 publicly available Affymetrix Genechip Citrus Genome microarray datasets, providing gene co-expression relationships at a genome-wide scale (33,000 transcripts). The comprehensive citrus GCN consists of a global GCN (condition-independent) and four condition-dependent GCNs that survey the sweet orange species only, all citrus fruit tissues, all citrus leaf tissues, or stress-exposed plants. All of these GCNs are clustered using genome-wide, gene-centric (guide) and graph clustering algorithms for flexibility of gene function prediction. For each putative cluster, gene ontology (GO) enrichment and gene expression specificity analyses were performed to enhance gene function, expression and regulation pattern prediction. The guide-gene approach was used to infer novel roles of genes involved in disease susceptibility and vitamin C metabolism, and graph-clustering approaches were used to investigate isoprenoid/phenylpropanoid metabolism in citrus peel, and citric acid catabolism via the GABA shunt in citrus fruit. Conclusions Integration of citrus gene co-expression networks

  14. Co-expression analysis identifies putative targets for CBP60g and SARD1 regulation

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    Truman William

    2012-11-01

    Full Text Available Abstract Background Salicylic acid is a critical signalling component in plant defence responses. In Arabidopsis, isochorismate synthase encoded by SID2 is essential for the biosynthesis of salicylic acid in response to biotic challenges. Recently, both the calmodulin binding protein CBP60g and its closest homolog, the non-calmodulin binding SARD1, have been shown to bind to the promoter region of SID2. Loss of both CBP60g and SARD1 severely impacts the plants ability to produce SA in response to bacterial inoculation and renders the plant susceptible to infection. In an electrophoretic mobility shift assay CBP60g and SARD1 were shown to bind specifically to a 10mer oligonucleotide with the sequence GAAATTTTGG. Results Gene expression profiling on a custom microarray identified a set of genes, like SID2, down-regulated in cbp60g sard1 mutant plants. Co-expression analysis across a defined set of ATH1 full genome microarray experiments expanded this gene set; clustering analysis was then applied to group densely interconnected genes. A stringent threshold for co-expression identified two related calmodulin-like genes tightly associated with SID2. SID2 was found to cluster with genes whose promoter regions were significantly enriched with GAAATT motifs. Genes clustering with SID2 were found to be down-regulated in the cbp60g sard1 double mutant. Representative genes from other clusters enriched with the GAAATT motif were found to be variously down-regulated, unchanged or up-regulated in the double mutant. A previously characterised co-expression between SID2 and WRKY28 was not reproduced in this analysis but was contained within a subset of the experiments where SID2 was co-expressed with CBP60g or SARD1. Conclusion Putative components of the CBP60g SARD1 signalling network have been uncovered by co-expression analysis. In addition to genes whose regulation is similar to that of SID2 some are repressed by CBP60g and SARD1.

  15. Purification and characterization of chaperonin 60 and heat-shock protein 70 from chromoplasts of Narcissus pseudonarcissus.

    Science.gov (United States)

    Bonk, M; Tadros, M; Vandekerckhove, J; Al-Babili, S; Beyer, P

    1996-01-01

    In chromoplast differentiation during flower formation in Narcissus pseudonarcissus, the molecular chaperones chaperonin 60 (Cpn60; alpha and beta) and heat-shock protein 70 (Hsp70) greatly increase in abundance. Both were purified and shown to be present in a functional form in chromoplasts, indicating their requirement in the extensive structural rearrangements during the chloroplast-to-chromoplast transition. The purified proteins, sequenced N terminally and from internal peptides, showed strong homology to plastid Cpn60 and Hsp 70 representatives from other plant species. During chromoplast differentiation, the carotenoid biosynthetic pathway is strongly induced. The corresponding enzymes are all nuclear encoded and form a large, soluble, hetero-oligomeric protein complex after import but prior to their membrane attachment. By immunoprecipitations we have shown that the plastid Hsp70 is a structural constituent of a soluble entity also containing phytoene desaturase. PMID:8754688

  16. Overexpression, purification, and characterization of beta-subunit group II chaperonin from hyperthermophilic Aeropyrum pernix K1.

    Science.gov (United States)

    Shin, Eun-Jung; Lee, Jin-Woo; Kim, Jeong-Hwan; Jeon, Sung-Jong; Kim, Yeon-Hee; Nam, Soo-Wan

    2010-03-01

    In the present study, overexpression, purification, and characterization of Aeropyrum pernix K1 chaperonin B in E. coli were investigated. The chaperonin beta-subunit gene (ApCpnB, 1,665 bp ORF) from the hyperthermophilic archaeon A. pernix K1 was amplified by PCR and subcloned into vector pET21a. The constructed pET21a-ApCpnB (6.9 kb) was transformed into E. coli BL21 Codonplus (DE3). The transformant cell successfully expressed ApCpnB, and the expression of ApCpnB (61.2 kDa) was identified through analysis of the fractions by SDS-PAGE (14% gel). The recombinant ApCpnB was purified to higher than 94% by using heat-shock treatment at 90 degrees C for 20 min and fast protein liquid chromatography on a HiTrap Q column step. The purified ApCpnB showed ATPase activity and its activity was dependent on temperature. In the presence of ATP, ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at 43 degrees C and 50 degrees C, respectively. Specifically, the activity of malate dehydrogenase (MDH) at 85 degrees C was greatly stabilized by the addition of ApCpnB and ATP. Coexpression of procarboxypeptidase B (pro-CPB) and ApCpnB in E. coli BL21 Codonplus (DE3) had a marked effect on the yield of pro-CPB as a soluble and active form, speculating that ApCpnB facilitates the correct folding of pro-CPB. These results suggest that ApCpnB has both foldase and holdase activities and can be used as a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.

  17. Functional characterization of the α- and β-subunits of a group II chaperonin from Aeropyrum pernix K1.

    Science.gov (United States)

    Lee, Jin-Woo; Kim, Se Won; Kim, Jeong-Hwan; Jeon, Sung-Jong; Kwon, Hyun-Ju; Kim, Byung-Woo; Nam, Soo-Wan

    2013-06-28

    We isolated and functionally characterized the α- and β- subunits (ApCpnA and ApCpnB) of a chaperonin from Aeropyrum pernix K1. The constructed vectors pET3d- ApCpnA and pET21a-ApCpnB were transformed into E. coli Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) cells. The expression of ApCpnA (60.7 kDa) and ApCpnB (61.2 kDa) was confirmed by SDS-PAGE analysis. Recombinant ApCpnA and ApCpnB were purified by heat-shock treatment and anion-exchange chromatography. ApCpnA and ApCpnB were able to hydrolyze not only ATP, but also CTP, GTP, and UTP, albeit with different efficacies. Purified ApCpnA and ApCpnB showed the highest ATPase, CTPase, UTPase, and GTPase activities at 80°C. Furthermore, the addition of ApCpnA and ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at 43°C and 50°C, respectively. In particular, the addition of ATP or CTP to ApCpnA and ApCpnB resulted in the most effective prevention of thermal aggregation and inactivation of CS and ADH. The ATPase activity of the two chaperonin subunits was dependent on the salt concentration. Among the ions we examined, potassium ions were the most effective at enhancing the ATP hydrolysis activity of ApCpnA and ApCpnB.

  18. Network statistics of genetically-driven gene co-expression modules in mouse crosses

    Directory of Open Access Journals (Sweden)

    Marie-Pier eScott-Boyer

    2013-12-01

    Full Text Available In biology, networks are used in different contexts as ways to represent relationships between entities, such as for instance interactions between genes, proteins or metabolites. Despite progress in the analysis of such networks and their potential to better understand the collective impact of genes on complex traits, one remaining challenge is to establish the biologic validity of gene co-expression networks and to determine what governs their organization. We used WGCNA to construct and analyze seven gene expression datasets from several tissues of mouse recombinant inbred strains (RIS. For six out of the 7 networks, we found that linkage to module QTLs (mQTLs could be established for 29.3% of gene co-expression modules detected in the several mouse RIS. For about 74.6% of such genetically-linked modules, the mQTL was on the same chromosome as the one contributing most genes to the module, with genes originating from that chromosome showing higher connectivity than other genes in the modules. Such modules (that we considered as genetically-driven had network statistic properties (density, centralization and heterogeneity that set them apart from other modules in the network. Altogether, a sizeable portion of gene co-expression modules detected in mouse RIS panels had genetic determinants as their main organizing principle. In addition to providing a biologic interpretation validation for these modules, these genetic determinants imparted on them particular properties that set them apart from other modules in the network, to the point that they can be predicted to a large extent on the basis of their network statistics.

  19. Co-expression network analysis and genetic algorithms for gene prioritization in preeclampsia.

    Science.gov (United States)

    Tejera, Eduardo; Bernardes, João; Rebelo, Irene

    2013-11-12

    In this study, we explored the gene prioritization in preeclampsia, combining co-expression network analysis and genetic algorithms optimization approaches. We analysed five public projects obtaining 1,146 significant genes after cross-platform and processing of 81 and 149 microarrays in preeclamptic and normal conditions, respectively. After co-expression network construction, modular and node analysis were performed using several approaches. Moreover, genetic algorithms were also applied in combination with the nearest neighbour and discriminant analysis classification methods. Significant differences were found in the genes connectivity distribution, both in normal and preeclampsia conditions pointing to the need and importance of examining connectivity alongside expression for prioritization. We discuss the global as well as intra-modular connectivity for hubs detection and also the utility of genetic algorithms in combination with the network information. FLT1, LEP, INHA and ENG genes were identified according to the literature, however, we also found other genes as FLNB, INHBA, NDRG1 and LYN highly significant but underexplored during normal pregnancy or preeclampsia. Weighted genes co-expression network analysis reveals a similar distribution along the modules detected both in normal and preeclampsia conditions. However, major differences were obtained by analysing the nodes connectivity. All models obtained by genetic algorithm procedures were consistent with a correct classification, higher than 90%, restricting to 30 variables in both classification methods applied.Combining the two methods we identified well known genes related to preeclampsia, but also lead us to propose new candidates poorly explored or completely unknown in the pathogenesis of preeclampsia, which may have to be validated experimentally.

  20. Conserved Units of Co-Expression in Bacterial Genomes: An Evolutionary Insight into Transcriptional Regulation

    Science.gov (United States)

    Junier, Ivan; Rivoire, Olivier

    2016-01-01

    Genome-wide measurements of transcriptional activity in bacteria indicate that the transcription of successive genes is strongly correlated beyond the scale of operons. Here, we analyze hundreds of bacterial genomes to identify supra-operonic segments of genes that are proximal in a large number of genomes. We show that these synteny segments correspond to genomic units of strong transcriptional co-expression. Structurally, the segments contain operons with specific relative orientations (co-directional or divergent) and nucleoid-associated proteins are found to bind at their boundaries. Functionally, operons inside a same segment are highly co-expressed even in the apparent absence of regulatory factors at their promoter regions. Remote operons along DNA can also be co-expressed if their corresponding segments share a transcriptional or sigma factor, without requiring these factors to bind directly to the promoters of the operons. As evidence that these results apply across the bacterial kingdom, we demonstrate them both in the Gram-negative bacterium Escherichia coli and in the Gram-positive bacterium Bacillus subtilis. The underlying process that we propose involves only RNA-polymerases and DNA: it implies that the transcription of an operon mechanically enhances the transcription of adjacent operons. In support of a primary role of this regulation by facilitated co-transcription, we show that the transcription en bloc of successive operons as a result of transcriptional read-through is strongly and specifically enhanced in synteny segments. Finally, our analysis indicates that facilitated co-transcription may be evolutionary primitive and may apply beyond bacteria. PMID:27195891

  1. Weighted gene co-expression network analysis of the peripheral blood from Amyotrophic Lateral Sclerosis patients

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    DeYoung Joseph

    2009-08-01

    Full Text Available Abstract Background Amyotrophic Lateral Sclerosis (ALS is a lethal disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord. Diagnosis is mainly based on clinical symptoms, and there is currently no therapy to stop the disease or slow its progression. Since access to spinal cord tissue is not possible at disease onset, we investigated changes in gene expression profiles in whole blood of ALS patients. Results Our transcriptional study showed dramatic changes in blood of ALS patients; 2,300 probes (9.4% showed significant differential expression in a discovery dataset consisting of 30 ALS patients and 30 healthy controls. Weighted gene co-expression network analysis (WGCNA was used to find disease-related networks (modules and disease related hub genes. Two large co-expression modules were found to be associated with ALS. Our findings were replicated in a second (30 patients and 30 controls and third dataset (63 patients and 63 controls, thereby demonstrating a highly significant and consistent association of two large co-expression modules with ALS disease status. Ingenuity Pathway Analysis of the ALS related module genes implicates enrichment of functional categories related to genetic disorders, neurodegeneration of the nervous system and inflammatory disease. The ALS related modules contain a number of candidate genes possibly involved in pathogenesis of ALS. Conclusion This first large-scale blood gene expression study in ALS observed distinct patterns between cases and controls which may provide opportunities for biomarker development as well as new insights into the molecular mechanisms of the disease.

  2. Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

    Science.gov (United States)

    Park, Jong-Won; Beyene, Getu; Buenrostro-Nava, Marco T.; Molina, Joe; Wang, Xiaofeng; Ciomperlik, Jessica J.; Manabayeva, Shuga A.; Alvarado, Veria Y.; Rathore, Keerti S.; Scholthof, Herman B.; Mirkov, T. Erik

    2013-01-01

    Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48–96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane. PMID:23799071

  3. Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies.

    Science.gov (United States)

    Romier, Christophe; Ben Jelloul, Marouane; Albeck, Shira; Buchwald, Gretel; Busso, Didier; Celie, Patrick H N; Christodoulou, Evangelos; De Marco, Valeria; van Gerwen, Suzan; Knipscheer, Puck; Lebbink, Joyce H; Notenboom, Valerie; Poterszman, Arnaud; Rochel, Natacha; Cohen, Serge X; Unger, Tamar; Sussman, Joel L; Moras, Dino; Sixma, Titia K; Perrakis, Anastassis

    2006-10-01

    Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells.

  4. Co-expression network analysis to identify pluripotency biomarkers in bovine and porcine embryos

    DEFF Research Database (Denmark)

    Mazzoni, Gianluca; Freude, Karla Kristine; Hall, Vanessa Jane

    the mouse data as proof of principle. In particular we studied cell populations from three different stages of pluripotency after fertilization: the inner cell mass, the epithelial epiblast and the gastrulating epiblast. Reads quality was checked with FASTQC, then the reads were pre-processed using Prinseq...... and mapped with STAR aligner ending up with a minimum of 80% of uniquely mapped reads per sample. Post mapping quality control with Qualimap showed a minimum of 60% of reads mapped in the exonic regions per sample. Finally the expression levels were estimated using HTSeq. Gene co-expression will be analyzed...

  5. Gene co-expression network analysis identifies porcine genes associated with variation in Salmonella shedding.

    Science.gov (United States)

    Kommadath, Arun; Bao, Hua; Arantes, Adriano S; Plastow, Graham S; Tuggle, Christopher K; Bearson, Shawn M D; Guan, Le Luo; Stothard, Paul

    2014-06-09

    Salmonella enterica serovar Typhimurium is a gram-negative bacterium that can colonise the gut of humans and several species of food producing farm animals to cause enteric or septicaemic salmonellosis. While many studies have looked into the host genetic response to Salmonella infection, relatively few have used correlation of shedding traits with gene expression patterns to identify genes whose variable expression among different individuals may be associated with differences in Salmonella clearance and resistance. Here, we aimed to identify porcine genes and gene co-expression networks that differentiate distinct responses to Salmonella challenge with respect to faecal Salmonella shedding. Peripheral blood transcriptome profiles from 16 pigs belonging to extremes of the trait of faecal Salmonella shedding counts recorded up to 20 days post-inoculation (low shedders (LS), n = 8; persistent shedders (PS), n = 8) were generated using RNA-sequencing from samples collected just before (day 0) and two days after (day 2) Salmonella inoculation. Weighted gene co-expression network analysis (WGCNA) of day 0 samples identified four modules of co-expressed genes significantly correlated with Salmonella shedding counts upon future challenge. Two of those modules consisted largely of innate immunity related genes, many of which were significantly up-regulated at day 2 post-inoculation. The connectivity at both days and the mean gene-wise expression levels at day 0 of the genes within these modules were higher in networks constructed using LS samples alone than those using PS alone. Genes within these modules include those previously reported to be involved in Salmonella resistance such as SLC11A1 (formerly NRAMP1), TLR4, CD14 and CCR1 and those for which an association with Salmonella is novel, for example, SIGLEC5, IGSF6 and TNFSF13B. Our analysis integrates gene co-expression network analysis, gene-trait correlations and differential expression to provide new

  6. Building gene co-expression networks using transcriptomics data for systems biology investigations

    DEFF Research Database (Denmark)

    Kadarmideen, Haja; Watson-Haigh, Nathan S.

    2012-01-01

    Gene co-expression networks (GCN), built using high-throughput gene expression data are fundamental aspects of systems biology. The main aims of this study were to compare two popular approaches to building and analysing GCN. We use real ovine microarray transcriptomics datasets representing four...... observed that, in contrast to WGCNA method, PCIT algorithm removes many of the edges of the most highly interconnected nodes. Removal of edges of most highly connected nodes or hub genes will have consequences for downstream analyses and biological interpretations. In general, for large GCN construction...

  7. Tissue and cell-type co-expression networks of transcription factors and wood component genes in Populus trichocarpa.

    Science.gov (United States)

    Shi, Rui; Wang, Jack P; Lin, Ying-Chung; Li, Quanzi; Sun, Ying-Hsuan; Chen, Hao; Sederoff, Ronald R; Chiang, Vincent L

    2017-05-01

    Co-expression networks based on transcriptomes of Populus trichocarpa major tissues and specific cell types suggest redundant control of cell wall component biosynthetic genes by transcription factors in wood formation. We analyzed the transcriptomes of five tissues (xylem, phloem, shoot, leaf, and root) and two wood forming cell types (fiber and vessel) of Populus trichocarpa to assemble gene co-expression subnetworks associated with wood formation. We identified 165 transcription factors (TFs) that showed xylem-, fiber-, and vessel-specific expression. Of these 165 TFs, 101 co-expressed (correlation coefficient, r > 0.7) with the 45 secondary cell wall cellulose, hemicellulose, and lignin biosynthetic genes. Each cell wall component gene co-expressed on average with 34 TFs, suggesting redundant control of the cell wall component gene expression. Co-expression analysis showed that the 101 TFs and the 45 cell wall component genes each has two distinct groups (groups 1 and 2), based on their co-expression patterns. The group 1 TFs (44 members) are predominantly xylem and fiber specific, and are all highly positively co-expressed with the group 1 cell wall component genes (30 members), suggesting their roles as major wood formation regulators. Group 1 TFs include a lateral organ boundary domain gene (LBD) that has the highest number of positively correlated cell wall component genes (36) and TFs (47). The group 2 TFs have 57 members, including 14 vessel-specific TFs, and are generally less correlated with the cell wall component genes. An exception is a vessel-specific basic helix-loop-helix (bHLH) gene that negatively correlates with 20 cell wall component genes, and may function as a key transcriptional suppressor. The co-expression networks revealed here suggest a well-structured transcriptional homeostasis for cell wall component biosynthesis during wood formation.

  8. Co-expression analysis reveals a group of genes potentially involved in regulation of plant response to iron-deficiency.

    Science.gov (United States)

    Li, Hua; Wang, Lei; Yang, Zhi Min

    2015-01-01

    Iron (Fe) is an essential element for plant growth and development. Iron deficiency results in abnormal metabolisms from respiration to photosynthesis. Exploration of Fe-deficient responsive genes and their networks is critically important to understand molecular mechanisms leading to the plant adaptation to soil Fe-limitation. Co-expression genes are a cluster of genes that have a similar expression pattern to execute relatively biological functions at a stage of development or under a certain environmental condition. They may share a common regulatory mechanism. In this study, we investigated Fe-starved-related co-expression genes from Arabidopsis. From the biological process GO annotation of TAIR (The Arabidopsis Information Resource), 180 iron-deficient responsive genes were detected. Using ATTED-II database, we generated six gene co-expression networks. Among these, two modules of PYE and IRT1 were successfully constructed. There are 30 co-expression genes that are incorporated in the two modules (12 in PYE-module and 18 in IRT1-module). Sixteen of the co-expression genes were well characterized. The remaining genes (14) are poorly or not functionally identified with iron stress. Validation of the 14 genes using real-time PCR showed differential expression under iron-deficiency. Most of the co-expression genes (23/30) could be validated in pye and fit mutant plants with iron-deficiency. We further identified iron-responsive cis-elements upstream of the co-expression genes and found that 22 out of 30 genes contain the iron-responsive motif IDE1. Furthermore, some auxin and ethylene-responsive elements were detected in the promoters of the co-expression genes. These results suggest that some of the genes can be also involved in iron stress response through the phytohormone-responsive pathways. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Once for All: A Novel Robust System for Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in Plants

    OpenAIRE

    Guitao Zhong; Qinlong Zhu; Yingxin Li; Yaoguang Liu; Hao Wang

    2017-01-01

    Chimeric fluorescent fusion proteins have been employed as a powerful tool to reveal the subcellular localizations and dynamics of proteins in living cells. Co-expression of a fluorescent fusion protein with well-known organelle markers in the same cell is especially useful in revealing its spatial and temporal functions of the protein in question. However, the conventional methods for co-expressing multiple fluorescent tagged proteins in plants have the drawbacks of low expression efficiency...

  10. Once for All: A Novel Robust System for Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in Plants

    Directory of Open Access Journals (Sweden)

    Guitao Zhong

    2017-06-01

    Full Text Available Chimeric fluorescent fusion proteins have been employed as a powerful tool to reveal the subcellular localizations and dynamics of proteins in living cells. Co-expression of a fluorescent fusion protein with well-known organelle markers in the same cell is especially useful in revealing its spatial and temporal functions of the protein in question. However, the conventional methods for co-expressing multiple fluorescent tagged proteins in plants have the drawbacks of low expression efficiency, variations in the expression level and time-consuming genetic crossing. Here, we have developed a novel robust system that allows for high-efficient co-expression of multiple chimeric fluorescent fusion proteins in plants in a time-saving fashion. This system takes advantage of employing a single expression vector which consists of multiple semi-independent expressing cassettes for the protein co-expression thereby overcoming the limitations of using multiple independent expressing plasmids. In addition, it is a highly manipulable DNA assembly system, in which modification and recombination of DNA molecules are easily achieved through an optimized one-step assembly reaction. By employing this effective system, we demonstrated that co-expression of two chimeric fluorescent fusion reporter proteins of vacuolar sorting receptor and secretory carrier membrane protein gave rise to their perspective subcellular localizations in plants via both transient expression and stable transformation. Thus, we believed that this technical advance represents a promising approach for multi-color-protein co-expression in plant cells.

  11. A contribution to the study of plant development evolution based on gene co-expression networks

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    Francisco J. Romero-Campero

    2013-08-01

    Full Text Available Phototrophic eukaryotes are among the most successful organisms on Earth due to their unparalleled efficiency at capturing light energy and fixing carbon dioxide to produce organic molecules. A conserved and efficient network of light-dependent regulatory modules could be at the bases of this success. This regulatory system conferred early advantages to phototrophic eukaryotes that allowed for specialization, complex developmental processes and modern plant characteristics. We have studied light-dependent gene regulatory modules from algae to plants employing integrative-omics approaches based on gene co-expression networks. Our study reveals some remarkably conserved ways in which eukaryotic phototrophs deal with day length and light signaling. Here we describe how a family of Arabidopsis transcription factors involved in photoperiod response has evolved from a single algal gene according to the innovation, amplification and divergence theory of gene evolution by duplication. These modifications of the gene co-expression networks from the ancient unicellular green algae Chlamydomonas reinhardtii to the modern brassica Arabidopsis thaliana may hint on the evolution and specialization of plants and other organisms.

  12. Disease association and inter-connectivity analysis of human brain specific co-expressed functional modules.

    Science.gov (United States)

    Oh, Kimin; Hwang, Taeho; Cha, Kihoon; Yi, Gwan-Su

    2015-12-16

    In the recent studies, it is suggested that the analysis of transcriptomic change of functional modules instead of individual genes would be more effective for system-wide identification of cellular functions. This could also provide a new possibility for the better understanding of difference between human and chimpanzee. In this study, we analyzed to find molecular characteristics of human brain functions from the difference of transcriptome between human and chimpanzee's brain using the functional module-centric co-expression analysis. We performed analysis of brain disease association and systems-level connectivity of species-specific co-expressed functional modules. Throughout the analyses, we found human-specific functional modules and significant overlap between their genes in known brain disease genes, suggesting that human brain disorder could be mediated by the perturbation of modular activities emerged in human brain specialization. In addition, the human-specific modules having neurobiological functions exhibited higher networking than other functional modules. This finding suggests that the expression of neural functions are more connected than other functions, and the resulting high-order brain functions could be identified as a result of consolidated inter-modular gene activities. Our result also showed that the functional module based transcriptome analysis has a potential to expand molecular understanding of high-order complex functions like cognitive abilities and brain disorders.

  13. Co-expression and co-purification of archaeal and eukaryal box C/D RNPs.

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    Yu Peng

    Full Text Available Box C/D ribonucleoprotein particles (RNPs are 2'-O-methylation enzymes required for maturation of ribosomal and small nuclear RNA. Previous biochemical and structural studies of the box C/D RNPs were limited by the unavailability of purified intact RNPs. We developed a bacterial co-expression strategy based on the combined use of a multi-gene expression system and a tRNA-scaffold construct that allowed the expression and purification of homogeneous archaeal and human box C/D RNPs. While the co-expressed and co-purified archaeal box C/D RNP was found to be fully active in a 2'-O-methylation assay, the intact human U14 box C/D RNP showed no detectable catalytic activity, consistent with the earlier findings that assembly of eukaryotic box C/D RNPs is nonspontaneous and requires additional protein factors. Our systems provide a means for further biochemical and structural characterization of box C/D RNPs and their assembly factors.

  14. Autocrine CSF-1 and CSF-1 Receptor Co-expression Promotes Renal Cell Carcinoma Growth

    Science.gov (United States)

    Menke, Julia; Kriegsmann, Jörg; Schimanski, Carl Christoph; Schwartz, Melvin M.; Schwarting, Andreas; Kelley, Vicki R.

    2011-01-01

    Renal cell carcinoma is increasing in incidence but the molecular mechanisms regulating its growth remain elusive. Co-expression of the monocytic growth factor CSF-1 and its receptor CSF-1R on renal tubular epithelial cells (TEC) will promote proliferation and anti-apoptosis during regeneration of renal tubules. Here we show that a CSF-1-dependent autocrine pathway is also responsible for the growth of renal cell carcinoma (RCC). CSF-1 and CSF-1R were co-expressed in RCC and TEC proximally adjacent to RCC. CSF-1 engagement of CSF-1R promoted RCC survival and proliferation and reduced apoptosis, in support of the likelihood that CSF-1R effector signals mediate RCC growth. In vivo CSF-1R blockade using a CSF-1R tyrosine kinase inhibitor decreased RCC proliferation and macrophage infiltration in a manner associated with a dramatic reduction in tumor mass. Further mechanistic investigations linked CSF-1 and EGF signaling in RCC. Taken together, our results suggest that budding RCC stimulates the proximal adjacent microenvironment in the kidney to release mediators of CSF-1, CSF-1R and EGF expression in RCC. Further, our findings imply that targeting CSF-1/CSF-1R signaling may be therapeutically effective in RCC. PMID:22052465

  15. Comparison of threshold selection methods for microarray gene co-expression matrices

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    Saxton Arnold M

    2009-12-01

    Full Text Available Abstract Background Network and clustering analyses of microarray co-expression correlation data often require application of a threshold to discard small correlations, thus reducing computational demands and decreasing the number of uninformative correlations. This study investigated threshold selection in the context of combinatorial network analysis of transcriptome data. Findings Six conceptually diverse methods - based on number of maximal cliques, correlation of control spots with expressed genes, top 1% of correlations, spectral graph clustering, Bonferroni correction of p-values, and statistical power - were used to estimate a correlation threshold for three time-series microarray datasets. The validity of thresholds was tested by comparison to thresholds derived from Gene Ontology information. Stability and reliability of the best methods were evaluated with block bootstrapping. Two threshold methods, number of maximal cliques and spectral graph, used information in the correlation matrix structure and performed well in terms of stability. Comparison to Gene Ontology found thresholds from number of maximal cliques extracted from a co-expression matrix were the most biologically valid. Approaches to improve both methods were suggested. Conclusion Threshold selection approaches based on network structure of gene relationships gave thresholds with greater relevance to curated biological relationships than approaches based on statistical pair-wise relationships.

  16. Strong correlation between ASPM gene expression and HCV cirrhosis progression identified by co-expression analysis.

    Science.gov (United States)

    Wang, Fan; Chang, Ying; Li, Jin; Wang, Hongling; Zhou, Rui; Qi, Jian; Liu, Jing; Zhao, Qiu

    2017-01-01

    Hepatitis C virus (HCV) cirrhosis is at a high risk of hepatocellular carcinoma (HCC), and its progression is influenced by a complex network of gene interactions. A weighted gene co-expression network was constructed to identify gene modules associated with the seven-stage disease progression from HCV cirrhosis to HCV-related HCC (n=65). In the significant module (R2=0.86), a total of 25 network hub genes were identified, half of which were also hub nodes in the protein-protein interaction network of the module genes. In validation, most hub genes showed a moderate correlation with the disease progression, and only ASPM was highly correlated (R2=0.801). In the test set (n=63), ASPM was also more highly expressed in HCV cirrhosis with concomitant HCC than in those without HCC (P=0.0054). Gene set enrichment analysis (GSEA) demonstrated that the gene set of "regulation of protein amino acid phosphorylation" (n=20) was enriched in HCV cirrhosis samples with ASPM highly expressed (false discovery rate (FDR)=0.049). In gene ontology (GO) analysis, genes in the enriched set were associated with liver neoplasms and other neoplastic diseases. In conclusion, through co-expression analysis, ASPM was identified and validated in association with the progression of HCV cirrhosis probably by regulating tumor-related phosphorylation. Copyright © 2016 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

  17. Discovery of Novel Human Gene Regulatory Modules from Gene Co-expression and Promoter Motif Analysis.

    Science.gov (United States)

    Ma, Shisong; Snyder, Michael; Dinesh-Kumar, Savithramma P

    2017-07-17

    Deciphering gene regulatory networks requires identification of gene expression modules. We describe a novel bottom-up approach to identify gene modules regulated by cis-regulatory motifs from a human gene co-expression network. Target genes of a cis-regulatory motif were identified from the network via the motif's enrichment or biased distribution towards transcription start sites in the promoters of co-expressed genes. A gene sub-network containing the target genes was extracted and used to derive gene modules. The analysis revealed known and novel gene modules regulated by the NF-Y motif. The binding of NF-Y proteins to these modules' gene promoters were verified using ENCODE ChIP-Seq data. The analyses also identified 8,048 Sp1 motif target genes, interestingly many of which were not detected by ENCODE ChIP-Seq. These target genes assemble into house-keeping, tissues-specific developmental, and immune response modules. Integration of Sp1 modules with genomic and epigenomic data indicates epigenetic control of Sp1 targets' expression in a cell/tissue specific manner. Finally, known and novel target genes and modules regulated by the YY1, RFX1, IRF1, and 34 other motifs were also identified. The study described here provides a valuable resource to understand transcriptional regulation of various human developmental, disease, or immunity pathways.

  18. Dynamic functional modules in co-expressed protein interaction networks of dilated cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Oyang Yen-Jen

    2010-10-01

    Full Text Available Abstract Background Molecular networks represent the backbone of molecular activity within cells and provide opportunities for understanding the mechanism of diseases. While protein-protein interaction data constitute static network maps, integration of condition-specific co-expression information provides clues to the dynamic features of these networks. Dilated cardiomyopathy is a leading cause of heart failure. Although previous studies have identified putative biomarkers or therapeutic targets for heart failure, the underlying molecular mechanism of dilated cardiomyopathy remains unclear. Results We developed a network-based comparative analysis approach that integrates protein-protein interactions with gene expression profiles and biological function annotations to reveal dynamic functional modules under different biological states. We found that hub proteins in condition-specific co-expressed protein interaction networks tended to be differentially expressed between biological states. Applying this method to a cohort of heart failure patients, we identified two functional modules that significantly emerged from the interaction networks. The dynamics of these modules between normal and disease states further suggest a potential molecular model of dilated cardiomyopathy. Conclusions We propose a novel framework to analyze the interaction networks in different biological states. It successfully reveals network modules closely related to heart failure; more importantly, these network dynamics provide new insights into the cause of dilated cardiomyopathy. The revealed molecular modules might be used as potential drug targets and provide new directions for heart failure therapy.

  19. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli.

    Science.gov (United States)

    Choi, Yoo Seong; Yang, Yun Jung; Yang, Byeongseon; Cha, Hyung Joon

    2012-10-24

    In nature, mussel adhesive proteins (MAPs) show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa) and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate the use of functional MAPs in practical applications and

  20. Genome-scale identification of cell-wall related genes in Arabidopsis based on co-expression network analysis

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    Wang Shan

    2012-08-01

    Full Text Available Abstract Background Identification of the novel genes relevant to plant cell-wall (PCW synthesis represents a highly important and challenging problem. Although substantial efforts have been invested into studying this problem, the vast majority of the PCW related genes remain unknown. Results Here we present a computational study focused on identification of the novel PCW genes in Arabidopsis based on the co-expression analyses of transcriptomic data collected under 351 conditions, using a bi-clustering technique. Our analysis identified 217 highly co-expressed gene clusters (modules under some experimental conditions, each containing at least one gene annotated as PCW related according to the Purdue Cell Wall Gene Families database. These co-expression modules cover 349 known/annotated PCW genes and 2,438 new candidates. For each candidate gene, we annotated the specific PCW synthesis stages in which it is involved and predicted the detailed function. In addition, for the co-expressed genes in each module, we predicted and analyzed their cis regulatory motifs in the promoters using our motif discovery pipeline, providing strong evidence that the genes in each co-expression module are transcriptionally co-regulated. From the all co-expression modules, we infer that 108 modules are related to four major PCW synthesis components, using three complementary methods. Conclusions We believe our approach and data presented here will be useful for further identification and characterization of PCW genes. All the predicted PCW genes, co-expression modules, motifs and their annotations are available at a web-based database: http://csbl.bmb.uga.edu/publications/materials/shanwang/CWRPdb/index.html.

  1. Mimicking the action of GroEL in molecular dynamics simulations : Application to the refinement of protein structures

    NARCIS (Netherlands)

    Fan, H; Mark, AE

    Bacterial chaperonin, GroEL, together with its co-chaperonin, GroES, facilitates the folding of a variety of polypeptides. Experiments suggest that GroEL stimulates protein folding by multiple cycles of binding and release. Misfolded proteins first bind to an exposed hydrophobic surface on GroEL.

  2. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

    Energy Technology Data Exchange (ETDEWEB)

    Bhaskar,; Kumari, Neeti [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India); Goyal, Neena, E-mail: neenacdri@yahoo.com [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer The study presents cloning and characterization of TCP1{gamma} gene from L. donovani. Black-Right-Pointing-Pointer TCP1{gamma} is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. Black-Right-Pointing-Pointer LdTCP{gamma} exhibited differential expression in different stages of promastigotes. Black-Right-Pointing-Pointer LdTCP{gamma} co-localized with actin, a cytoskeleton protein. Black-Right-Pointing-Pointer The data suggests that this gene may have a role in differentiation/biogenesis. Black-Right-Pointing-Pointer First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1{gamma}), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1{gamma} of Leishmania donovani (LdTCP1{gamma}), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1{gamma} revealed the presence of all the characteristic features of TCP1{gamma}. However, leishmanial TCP1{gamma} represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1{gamma} exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1{gamma} as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1{gamma} was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1{gamma} with actin suggests

  3. Gene co-expression networks and profiles reveal potential biomarkers of boar taint in pigs

    DEFF Research Database (Denmark)

    Drag, Markus; Skinkyté-Juskiené, R.; Do, D. N.

    and enrichment analysis and semantic filtering revealed the GO terms “catalytic activity” and “transferase activity” to be overrepresented (p steroid hormones. Extraction of hub...... active tissue. GOseq was used to find enriched gene ontology (GO) terms and REVIGO was used to filter semantic similarities. In both liver and testis, a GO termed “oxidoreductase activity” was enriched (p steroid metabolism, including androstenone...... synthesis. In testis, >80 DE genes were functionally classified by the PANTHER tool to “Gonadotropin releasing hormone receptor” and “Wnt signaling” pathways which play a role in reproductive maturation and proliferation of spermatogonia, respectively. WGCNA was used to build co-expression modules...

  4. Computational gene expression profiling under salt stress reveals patterns of co-expression.

    Science.gov (United States)

    Sanchita; Sharma, Ashok

    2016-03-01

    Plants respond differently to environmental conditions. Among various abiotic stresses, salt stress is a condition where excess salt in soil causes inhibition of plant growth. To understand the response of plants to the stress conditions, identification of the responsible genes is required. Clustering is a data mining technique used to group the genes with similar expression. The genes of a cluster show similar expression and function. We applied clustering algorithms on gene expression data of Solanum tuberosum showing differential expression in Capsicum annuum under salt stress. The clusters, which were common in multiple algorithms were taken further for analysis. Principal component analysis (PCA) further validated the findings of other cluster algorithms by visualizing their clusters in three-dimensional space. Functional annotation results revealed that most of the genes were involved in stress related responses. Our findings suggest that these algorithms may be helpful in the prediction of the function of co-expressed genes.

  5. Computational gene expression profiling under salt stress reveals patterns of co-expression

    Directory of Open Access Journals (Sweden)

    Sanchita

    2016-03-01

    Full Text Available Plants respond differently to environmental conditions. Among various abiotic stresses, salt stress is a condition where excess salt in soil causes inhibition of plant growth. To understand the response of plants to the stress conditions, identification of the responsible genes is required. Clustering is a data mining technique used to group the genes with similar expression. The genes of a cluster show similar expression and function. We applied clustering algorithms on gene expression data of Solanum tuberosum showing differential expression in Capsicum annuum under salt stress. The clusters, which were common in multiple algorithms were taken further for analysis. Principal component analysis (PCA further validated the findings of other cluster algorithms by visualizing their clusters in three-dimensional space. Functional annotation results revealed that most of the genes were involved in stress related responses. Our findings suggest that these algorithms may be helpful in the prediction of the function of co-expressed genes.

  6. Protein-protein interaction and gene co-expression maps of ARFs and Aux/IAAs in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Sarbottam ePiya

    2014-12-01

    Full Text Available The phytohormone auxin regulates nearly all aspects of plant growth and development. Based on the current model in Arabidopsis thaliana, Auxin/indole-3-acetic acid (Aux/IAA proteins repress auxin-inducible genes by inhibiting auxin response transcription factors (ARFs. Experimental evidence suggests that heterodimerization between Aux/IAA and ARF proteins are related to their unique biological functions. The objective of this study was to generate the Aux/IAA-ARF protein-protein interaction map using full length sequences and locate the interacting protein pairs to specific gene co-expression networks in order to define tissue-specific responses of the Aux/IAA-ARF interactome. Pairwise interactions between 19 ARFs and 29 Aux/IAAs resulted in the identification of 213 specific interactions of which 79 interactions were previously unknown. The incorporation of co-expression profiles with protein-protein interaction data revealed a strong correlation of gene co-expression for 70% of the ARF-Aux/IAA interacting pairs in at least one tissue/organ, indicative of the biological significance of these interactions. Importantly, ARF4-8 and 19, which were found to interact with almost all Aux-Aux/IAA showed broad co-expression relationships with Aux/IAA genes, thus, formed the central hubs of the co-expression network. Our analyses provide new insights into the biological significance of ARF-Aux/IAA associations in the morphogenesis and development of various plant tissues and organs.

  7. Protein-protein interaction and gene co-expression maps of ARFs and Aux/IAAs in Arabidopsis.

    Science.gov (United States)

    Piya, Sarbottam; Shrestha, Sandesh K; Binder, Brad; Stewart, C Neal; Hewezi, Tarek

    2014-01-01

    The phytohormone auxin regulates nearly all aspects of plant growth and development. Based on the current model in Arabidopsis thaliana, Auxin/indole-3-acetic acid (Aux/IAA) proteins repress auxin-inducible genes by inhibiting auxin response transcription factors (ARFs). Experimental evidence suggests that heterodimerization between Aux/IAA and ARF proteins are related to their unique biological functions. The objective of this study was to generate the Aux/IAA-ARF protein-protein interaction map using full length sequences and locate the interacting protein pairs to specific gene co-expression networks in order to define tissue-specific responses of the Aux/IAA-ARF interactome. Pairwise interactions between 19 ARFs and 29 Aux/IAAs resulted in the identification of 213 specific interactions of which 79 interactions were previously unknown. The incorporation of co-expression profiles with protein-protein interaction data revealed a strong correlation of gene co-expression for 70% of the ARF-Aux/IAA interacting pairs in at least one tissue/organ, indicative of the biological significance of these interactions. Importantly, ARF4-8 and 19, which were found to interact with almost all Aux-Aux/IAA showed broad co-expression relationships with Aux/IAA genes, thus, formed the central hubs of the co-expression network. Our analyses provide new insights into the biological significance of ARF-Aux/IAA associations in the morphogenesis and development of various plant tissues and organs.

  8. Integrated Weighted Gene Co-expression Network Analysis with an Application to Chronic Fatigue Syndrome

    Directory of Open Access Journals (Sweden)

    Rajeevan Mangalathu S

    2008-11-01

    Full Text Available Abstract Background Systems biologic approaches such as Weighted Gene Co-expression Network Analysis (WGCNA can effectively integrate gene expression and trait data to identify pathways and candidate biomarkers. Here we show that the additional inclusion of genetic marker data allows one to characterize network relationships as causal or reactive in a chronic fatigue syndrome (CFS data set. Results We combine WGCNA with genetic marker data to identify a disease-related pathway and its causal drivers, an analysis which we refer to as "Integrated WGCNA" or IWGCNA. Specifically, we present the following IWGCNA approach: 1 construct a co-expression network, 2 identify trait-related modules within the network, 3 use a trait-related genetic marker to prioritize genes within the module, 4 apply an integrated gene screening strategy to identify candidate genes and 5 carry out causality testing to verify and/or prioritize results. By applying this strategy to a CFS data set consisting of microarray, SNP and clinical trait data, we identify a module of 299 highly correlated genes that is associated with CFS severity. Our integrated gene screening strategy results in 20 candidate genes. We show that our approach yields biologically interesting genes that function in the same pathway and are causal drivers for their parent module. We use a separate data set to replicate findings and use Ingenuity Pathways Analysis software to functionally annotate the candidate gene pathways. Conclusion We show how WGCNA can be combined with genetic marker data to identify disease-related pathways and the causal drivers within them. The systems genetics approach described here can easily be used to generate testable genetic hypotheses in other complex disease studies.

  9. Estimation of the proteomic cancer co-expression sub networks by using association estimators.

    Science.gov (United States)

    Erdoğan, Cihat; Kurt, Zeyneb; Diri, Banu

    2017-01-01

    In this study, the association estimators, which have significant influences on the gene network inference methods and used for determining the molecular interactions, were examined within the co-expression network inference concept. By using the proteomic data from five different cancer types, the hub genes/proteins within the disease-associated gene-gene/protein-protein interaction sub networks were identified. Proteomic data from various cancer types is collected from The Cancer Proteome Atlas (TCPA). Correlation and mutual information (MI) based nine association estimators that are commonly used in the literature, were compared in this study. As the gold standard to measure the association estimators' performance, a multi-layer data integration platform on gene-disease associations (DisGeNET) and the Molecular Signatures Database (MSigDB) was used. Fisher's exact test was used to evaluate the performance of the association estimators by comparing the created co-expression networks with the disease-associated pathways. It was observed that the MI based estimators provided more successful results than the Pearson and Spearman correlation approaches, which are used in the estimation of biological networks in the weighted correlation network analysis (WGCNA) package. In correlation-based methods, the best average success rate for five cancer types was 60%, while in MI-based methods the average success ratio was 71% for James-Stein Shrinkage (Shrink) and 64% for Schurmann-Grassberger (SG) association estimator, respectively. Moreover, the hub genes and the inferred sub networks are presented for the consideration of researchers and experimentalists.

  10. Estimation of the proteomic cancer co-expression sub networks by using association estimators.

    Directory of Open Access Journals (Sweden)

    Cihat Erdoğan

    Full Text Available In this study, the association estimators, which have significant influences on the gene network inference methods and used for determining the molecular interactions, were examined within the co-expression network inference concept. By using the proteomic data from five different cancer types, the hub genes/proteins within the disease-associated gene-gene/protein-protein interaction sub networks were identified. Proteomic data from various cancer types is collected from The Cancer Proteome Atlas (TCPA. Correlation and mutual information (MI based nine association estimators that are commonly used in the literature, were compared in this study. As the gold standard to measure the association estimators' performance, a multi-layer data integration platform on gene-disease associations (DisGeNET and the Molecular Signatures Database (MSigDB was used. Fisher's exact test was used to evaluate the performance of the association estimators by comparing the created co-expression networks with the disease-associated pathways. It was observed that the MI based estimators provided more successful results than the Pearson and Spearman correlation approaches, which are used in the estimation of biological networks in the weighted correlation network analysis (WGCNA package. In correlation-based methods, the best average success rate for five cancer types was 60%, while in MI-based methods the average success ratio was 71% for James-Stein Shrinkage (Shrink and 64% for Schurmann-Grassberger (SG association estimator, respectively. Moreover, the hub genes and the inferred sub networks are presented for the consideration of researchers and experimentalists.

  11. Identifying key genes in rheumatoid arthritis by weighted gene co-expression network analysis.

    Science.gov (United States)

    Ma, Chunhui; Lv, Qi; Teng, Songsong; Yu, Yinxian; Niu, Kerun; Yi, Chengqin

    2017-08-01

    This study aimed to identify rheumatoid arthritis (RA) related genes based on microarray data using the WGCNA (weighted gene co-expression network analysis) method. Two gene expression profile datasets GSE55235 (10 RA samples and 10 healthy controls) and GSE77298 (16 RA samples and seven healthy controls) were downloaded from Gene Expression Omnibus database. Characteristic genes were identified using metaDE package. WGCNA was used to find disease-related networks based on gene expression correlation coefficients, and module significance was defined as the average gene significance of all genes used to assess the correlation between the module and RA status. Genes in the disease-related gene co-expression network were subject to functional annotation and pathway enrichment analysis using Database for Annotation Visualization and Integrated Discovery. Characteristic genes were also mapped to the Connectivity Map to screen small molecules. A total of 599 characteristic genes were identified. For each dataset, characteristic genes in the green, red and turquoise modules were most closely associated with RA, with gene numbers of 54, 43 and 79, respectively. These genes were enriched in totally enriched in 17 Gene Ontology terms, mainly related to immune response (CD97, FYB, CXCL1, IKBKE, CCR1, etc.), inflammatory response (CD97, CXCL1, C3AR1, CCR1, LYZ, etc.) and homeostasis (C3AR1, CCR1, PLN, CCL19, PPT1, etc.). Two small-molecule drugs sanguinarine and papaverine were predicted to have a therapeutic effect against RA. Genes related to immune response, inflammatory response and homeostasis presumably have critical roles in RA pathogenesis. Sanguinarine and papaverine have a potential therapeutic effect against RA. © 2017 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.

  12. Comparative analysis of weighted gene co-expression networks in human and mouse.

    Science.gov (United States)

    Eidsaa, Marius; Stubbs, Lisa; Almaas, Eivind

    2017-01-01

    The application of complex network modeling to analyze large co-expression data sets has gained traction during the last decade. In particular, the use of the weighted gene co-expression network analysis framework has allowed an unbiased and systems-level investigation of genotype-phenotype relationships in a wide range of systems. Since mouse is an important model organism for biomedical research on human disease, it is of great interest to identify similarities and differences in the functional roles of human and mouse orthologous genes. Here, we develop a novel network comparison approach which we demonstrate by comparing two gene-expression data sets from a large number of human and mouse tissues. The method uses weighted topological overlap alongside the recently developed network-decomposition method of s-core analysis, which is suitable for making gene-centrality rankings for weighted networks. The aim is to identify globally central genes separately in the human and mouse networks. By comparing the ranked gene lists, we identify genes that display conserved or diverged centrality-characteristics across the networks. This framework only assumes a single threshold value that is chosen from a statistical analysis, and it may be applied to arbitrary network structures and edge-weight distributions, also outside the context of biology. When conducting the comparative network analysis, both within and across the two species, we find a clear pattern of enrichment of transcription factors, for the homeobox domain in particular, among the globally central genes. We also perform gene-ontology term enrichment analysis and look at disease-related genes for the separate networks as well as the network comparisons. We find that gene ontology terms related to regulation and development are generally enriched across the networks. In particular, the genes FOXE3, RHO, RUNX2, ALX3 and RARA, which are disease genes in either human or mouse, are on the top-10 list of globally

  13. Integrated weighted gene co-expression network analysis with an application to chronic fatigue syndrome.

    Science.gov (United States)

    Presson, Angela P; Sobel, Eric M; Papp, Jeanette C; Suarez, Charlyn J; Whistler, Toni; Rajeevan, Mangalathu S; Vernon, Suzanne D; Horvath, Steve

    2008-11-06

    Systems biologic approaches such as Weighted Gene Co-expression Network Analysis (WGCNA) can effectively integrate gene expression and trait data to identify pathways and candidate biomarkers. Here we show that the additional inclusion of genetic marker data allows one to characterize network relationships as causal or reactive in a chronic fatigue syndrome (CFS) data set. We combine WGCNA with genetic marker data to identify a disease-related pathway and its causal drivers, an analysis which we refer to as "Integrated WGCNA" or IWGCNA. Specifically, we present the following IWGCNA approach: 1) construct a co-expression network, 2) identify trait-related modules within the network, 3) use a trait-related genetic marker to prioritize genes within the module, 4) apply an integrated gene screening strategy to identify candidate genes and 5) carry out causality testing to verify and/or prioritize results. By applying this strategy to a CFS data set consisting of microarray, SNP and clinical trait data, we identify a module of 299 highly correlated genes that is associated with CFS severity. Our integrated gene screening strategy results in 20 candidate genes. We show that our approach yields biologically interesting genes that function in the same pathway and are causal drivers for their parent module. We use a separate data set to replicate findings and use Ingenuity Pathways Analysis software to functionally annotate the candidate gene pathways. We show how WGCNA can be combined with genetic marker data to identify disease-related pathways and the causal drivers within them. The systems genetics approach described here can easily be used to generate testable genetic hypotheses in other complex disease studies.

  14. The chaperonin containing TCP1 complex (CCT/TRiC) is involved in mediating sperm-oocyte interaction.

    Science.gov (United States)

    Dun, Matthew D; Smith, Nathan D; Baker, Mark A; Lin, Minjie; Aitken, R John; Nixon, Brett

    2011-10-21

    Sperm-oocyte interactions are among the most remarkable processes in cell biology. These cellular recognition events are initiated by an exquisitely specific adhesion of free-swimming spermatozoa to the zona pellucida, an acellular matrix that surrounds the ovulated oocyte. Decades of research focusing on this interaction have led to the establishment of a widely held paradigm that the zona pellucida receptor is a single molecular entity that is constitutively expressed on the sperm cell surface. In contrast, we have employed the techniques of blue native-polyacrylamide gel electrophoresis, far Western blotting, and proximity ligation to secure the first direct evidence in support of a novel hypothesis that zona binding is mediated by multimeric sperm receptor complex(es). Furthermore, we show that one such multimeric association, comprising the chaperonin-containing TCP1 complex (CCT/TRiC) and a zona-binding protein, zona pellucida-binding protein 2, is present on the surface of capacitated spermatozoa and could account for the zona binding activity of these cells. Collectively, these data provide an important biochemical insight into the molecular basis of sperm-zona pellucida interaction and a plausible explanation for how spermatozoa gain their ability to fertilize.

  15. ComPlEx: conservation and divergence of co-expression networks in A. thaliana, Populus and O. sativa.

    Science.gov (United States)

    Netotea, Sergiu; Sundell, David; Street, Nathaniel R; Hvidsten, Torgeir R

    2014-02-06

    Divergence in gene regulation has emerged as a key mechanism underlying species differentiation. Comparative analysis of co-expression networks across species can reveal conservation and divergence in the regulation of genes. We inferred co-expression networks of A. thaliana, Populus spp. and O. sativa using state-of-the-art methods based on mutual information and context likelihood of relatedness, and conducted a comprehensive comparison of these networks across a range of co-expression thresholds. In addition to quantifying gene-gene link and network neighbourhood conservation, we also applied recent advancements in network analysis to do cross-species comparisons of network properties such as scale free characteristics and gene centrality as well as network motifs. We found that in all species the networks emerged as scale free only above a certain co-expression threshold, and that the high-centrality genes upholding this organization tended to be conserved. Network motifs, in particular the feed-forward loop, were found to be significantly enriched in specific functional subnetworks but where much less conserved across species than gene centrality. Although individual gene-gene co-expression had massively diverged, up to ~80% of the genes still had a significantly conserved network neighbourhood. For genes with multiple predicted orthologs, about half had one ortholog with conserved regulation and another ortholog with diverged or non-conserved regulation. Furthermore, the most sequence similar ortholog was not the one with the most conserved gene regulation in over half of the cases. We have provided a comprehensive analysis of gene regulation evolution in plants and built a web tool for Comparative analysis of Plant co-Expression networks (ComPlEx, http://complex.plantgenie.org/). The tool can be particularly useful for identifying the ortholog with the most conserved regulation among several sequence-similar alternatives and can thus be of practical

  16. Evaluation and improvement of the regulatory inference for large co-expression networks with limited sample size.

    Science.gov (United States)

    Guo, Wenbin; Calixto, Cristiane P G; Tzioutziou, Nikoleta; Lin, Ping; Waugh, Robbie; Brown, John W S; Zhang, Runxuan

    2017-06-19

    Co-expression has been widely used to identify novel regulatory relationships using high throughput measurements, such as microarray and RNA-seq data. Evaluation studies on co-expression network analysis methods mostly focus on networks of small or medium size of up to a few hundred nodes. For large networks, simulated expression data usually consist of hundreds or thousands of profiles with different perturbations or knock-outs, which is uncommon in real experiments due to their cost and the amount of work required. Thus, the performances of co-expression network analysis methods on large co-expression networks consisting of a few thousand nodes, with only a small number of profiles with a single perturbation, which more accurately reflect normal experimental conditions, are generally uncharacterized and unknown. We proposed a novel network inference methods based on Relevance Low order Partial Correlation (RLowPC). RLowPC method uses a two-step approach to select on the high-confidence edges first by reducing the search space by only picking the top ranked genes from an intial partial correlation analysis and, then computes the partial correlations in the confined search space by only removing the linear dependencies from the shared neighbours, largely ignoring the genes showing lower association. We selected six co-expression-based methods with good performance in evaluation studies from the literature: Partial correlation, PCIT, ARACNE, MRNET, MRNETB and CLR. The evaluation of these methods was carried out on simulated time-series data with various network sizes ranging from 100 to 3000 nodes. Simulation results show low precision and recall for all of the above methods for large networks with a small number of expression profiles. We improved the inference significantly by refinement of the top weighted edges in the pre-inferred partial correlation networks using RLowPC. We found improved performance by partitioning large networks into smaller co-expressed

  17. Meta-analysis of inter-species liver co-expression networks elucidates traits associated with common human diseases.

    Directory of Open Access Journals (Sweden)

    Kai Wang

    2009-12-01

    Full Text Available Co-expression networks are routinely used to study human diseases like obesity and diabetes. Systematic comparison of these networks between species has the potential to elucidate common mechanisms that are conserved between human and rodent species, as well as those that are species-specific characterizing evolutionary plasticity. We developed a semi-parametric meta-analysis approach for combining gene-gene co-expression relationships across expression profile datasets from multiple species. The simulation results showed that the semi-parametric method is robust against noise. When applied to human, mouse, and rat liver co-expression networks, our method out-performed existing methods in identifying gene pairs with coherent biological functions. We identified a network conserved across species that highlighted cell-cell signaling, cell-adhesion and sterol biosynthesis as main biological processes represented in genome-wide association study candidate gene sets for blood lipid levels. We further developed a heterogeneity statistic to test for network differences among multiple datasets, and demonstrated that genes with species-specific interactions tend to be under positive selection throughout evolution. Finally, we identified a human-specific sub-network regulated by RXRG, which has been validated to play a different role in hyperlipidemia and Type 2 diabetes between human and mouse. Taken together, our approach represents a novel step forward in integrating gene co-expression networks from multiple large scale datasets to leverage not only common information but also differences that are dataset-specific.

  18. CoGA: An R Package to Identify Differentially Co-Expressed Gene Sets by Analyzing the Graph Spectra.

    Directory of Open Access Journals (Sweden)

    Suzana de Siqueira Santos

    Full Text Available Gene set analysis aims to identify predefined sets of functionally related genes that are differentially expressed between two conditions. Although gene set analysis has been very successful, by incorporating biological knowledge about the gene sets and enhancing statistical power over gene-by-gene analyses, it does not take into account the correlation (association structure among the genes. In this work, we present CoGA (Co-expression Graph Analyzer, an R package for the identification of groups of differentially associated genes between two phenotypes. The analysis is based on concepts of Information Theory applied to the spectral distributions of the gene co-expression graphs, such as the spectral entropy to measure the randomness of a graph structure and the Jensen-Shannon divergence to discriminate classes of graphs. The package also includes common measures to compare gene co-expression networks in terms of their structural properties, such as centrality, degree distribution, shortest path length, and clustering coefficient. Besides the structural analyses, CoGA also includes graphical interfaces for visual inspection of the networks, ranking of genes according to their "importance" in the network, and the standard differential expression analysis. We show by both simulation experiments and analyses of real data that the statistical tests performed by CoGA indeed control the rate of false positives and is able to identify differentially co-expressed genes that other methods failed.

  19. CoGA: An R Package to Identify Differentially Co-Expressed Gene Sets by Analyzing the Graph Spectra.

    Science.gov (United States)

    Santos, Suzana de Siqueira; Galatro, Thais Fernanda de Almeida; Watanabe, Rodrigo Akira; Oba-Shinjo, Sueli Mieko; Nagahashi Marie, Suely Kazue; Fujita, André

    2015-01-01

    Gene set analysis aims to identify predefined sets of functionally related genes that are differentially expressed between two conditions. Although gene set analysis has been very successful, by incorporating biological knowledge about the gene sets and enhancing statistical power over gene-by-gene analyses, it does not take into account the correlation (association) structure among the genes. In this work, we present CoGA (Co-expression Graph Analyzer), an R package for the identification of groups of differentially associated genes between two phenotypes. The analysis is based on concepts of Information Theory applied to the spectral distributions of the gene co-expression graphs, such as the spectral entropy to measure the randomness of a graph structure and the Jensen-Shannon divergence to discriminate classes of graphs. The package also includes common measures to compare gene co-expression networks in terms of their structural properties, such as centrality, degree distribution, shortest path length, and clustering coefficient. Besides the structural analyses, CoGA also includes graphical interfaces for visual inspection of the networks, ranking of genes according to their "importance" in the network, and the standard differential expression analysis. We show by both simulation experiments and analyses of real data that the statistical tests performed by CoGA indeed control the rate of false positives and is able to identify differentially co-expressed genes that other methods failed.

  20. Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system

    Directory of Open Access Journals (Sweden)

    Lalonde Sylvie

    2003-03-01

    Full Text Available Abstract Background The Arabidopsis genome contains nine sucrose transporter paralogs falling into three clades: SUT1-like, SUT2 and SUT4. The carriers differ in their kinetic properties. Many transport proteins are known to exist as oligomers. The yeast-based split ubiquitin system can be used to analyze the ability of membrane proteins to interact. Results Promoter-GUS fusions were used to analyze the cellular expression of the three transporter genes in transgenic Arabidopsis plants. All three fusion genes are co-expressed in companion cells. Protein-protein interactions between Arabidopsis sucrose transporters were tested using the split ubiquitin system. Three paralogous sucrose transporters are capable of interacting as either homo- or heteromers. The interactions are specific, since a potassium channel and a glucose transporter did not show interaction with sucrose transporters. Also the biosynthetic and metabolizing enzymes, sucrose phosphate phosphatase and sucrose synthase, which were found to be at least in part bound to the plasma membrane, did not specifically interact with sucrose transporters. Conclusions The split-ubiquitin system provides a powerful tool to detect potential interactions between plant membrane proteins by heterologous expression in yeast, and can be used to screen for interactions with membrane proteins as baits. Like other membrane proteins, the Arabidopsis sucrose transporters are able to form oligomers. The biochemical approaches are required to confirm the in planta interaction.

  1. clc is co-expressed with clf or cntfr in developing mouse muscles

    Directory of Open Access Journals (Sweden)

    Gascan Hugues

    2005-01-01

    Full Text Available Abstract Background The ciliary neurotrophic factor (CNTF receptor is composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor, associated with a non-signalling CNTF binding receptor α component (CNTFR. This tripartite receptor has been shown to play important roles in the development of motor neurons, but the identity of the relevant ligand(s is still not clearly established. Recently, we have identified two new ligands for the CNTF receptor complex. These are heterodimeric cytokines composed of cardiotrophin-like cytokine (CLC associated either with the soluble receptor subunit cytokine-like factor-1 (CLF or the soluble form of the binding receptor itself (sCNTFR. Results Here we show that, during development, clc is expressed in lung, kidney, vibrissae, tooth, epithelia and muscles during the period of development corresponding to when motoneuron loss is observed in mice lacking a functional CNTF receptor. In addition, we demonstrate that it is co-expressed at the single cell level with clf and cntfr, supporting the idea that CLC might be co-secreted with either CLF or sCNTFR. Conclusion This expression pattern is in favor of CLC, associated either with CLF or sCNTFR, being an important player in the signal triggered by the CNTF receptor being required for motoneuron development.

  2. Co-expression and characterization of enterocin CRL35 and its mutant in Escherichia coli Rosetta

    Directory of Open Access Journals (Sweden)

    Masías Emilse

    2014-01-01

    Full Text Available Even though many sequences and structures of bacteriocins from lactic acid bacteria have been fully characterized so far, little information is currently available about bacteriocins heterologously produced by Escherichia coli. For this purpose, the structural gene of enterocin CRL35, munA, was PCR-amplified using specific primers and cloned downstream of PelB sequence in the pET22b (+ expression vector. E. coli Rosetta (DE3 pLysS was chosen as the host for production and enterocin was purified by an easy two-step protocol. The bacteriocin was correctly expressed with the expected intramolecular disulfide bond. Nevertheless, it was found that a variant of the enterocin, differing by 12 Da from the native polypeptide, was co-expressed by E. coli Rosetta in comparable amount. Indeed, the mutant bacteriocin contained two amino acid substitutions that were characterized by matrix assisted laser desorption ionization-time of flight (MALDI-TOF and HPLCelectrospray (ESI-Q-TOF tandem mass spectrometry (MS/ MS sequencing. This is the first report regarding the production of mutants of pediocin-like bacteriocins in the E. coli expression system.

  3. MIDBRAIN CATECHOLAMINERGIC NEURONS CO-EXPRESS α-SYNUCLEIN AND TAU IN PROGRESSIVE SUPRANUCLEAR PALSY

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    María Elena eErro Aguirre

    2015-03-01

    Full Text Available Objective: To analyze the frequency and distribution of α-synuclein deposits in progressive supranuclear palsy (PSP.Methods: The brains of 25 cases of pathologically confirmed PSP were evaluated with immunohistochemistry for α-synuclein and tau. Multiple immunofluorescent stains were applied to analyze the expression of tau and α-synuclein aggregates in catecholaminergic neurons. Patients’ clinical symptoms were retrospectively recorded. Results: Deposits α-synuclein in the form of typical Lewy bodies (LBs were only found in two PSP cases (8% that fulfilled the clinical subtype of PSP known as Richardson’s syndrome (RS. LBs were present in the locus ceruleus, substantia nigra pars compacta, basal forebrain, amygdala and cingulated cortex in a distribution mimicking that of Parkinson’s disease. Triple-immunolabeling revealed co-expression of α-synuclein and tau proteins in some tyrosine hydroxilase-positive neurons of the locus ceruleus and substantia nigra pars compacta.Conclusions: There is no apparent clinical correlation between the presence of LBs in PSP. Tau protein co-aggregate with α-synuclein in catecholaminergic neurons of PSP brains suggesting a synergistic interaction between the two proteins. This is in keeping with the current view of neurodegenerative disorders as ‘misfolded protein diseases’.

  4. Genetic Network Inference: From Co-Expression Clustering to Reverse Engineering

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    Dhaeseleer, Patrik; Liang, Shoudan; Somogyi, Roland

    2000-01-01

    Advances in molecular biological, analytical, and computational technologies are enabling us to systematically investigate the complex molecular processes underlying biological systems. In particular, using high-throughput gene expression assays, we are able to measure the output of the gene regulatory network. We aim here to review datamining and modeling approaches for conceptualizing and unraveling the functional relationships implicit in these datasets. Clustering of co-expression profiles allows us to infer shared regulatory inputs and functional pathways. We discuss various aspects of clustering, ranging from distance measures to clustering algorithms and multiple-duster memberships. More advanced analysis aims to infer causal connections between genes directly, i.e., who is regulating whom and how. We discuss several approaches to the problem of reverse engineering of genetic networks, from discrete Boolean networks, to continuous linear and non-linear models. We conclude that the combination of predictive modeling with systematic experimental verification will be required to gain a deeper insight into living organisms, therapeutic targeting, and bioengineering.

  5. Construction and comparison of gene co-expression networks shows complex plant immune responses

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    Luis Guillermo Leal

    2014-10-01

    Full Text Available Gene co-expression networks (GCNs are graphic representations that depict the coordinated transcription of genes in response to certain stimuli. GCNs provide functional annotations of genes whose function is unknown and are further used in studies of translational functional genomics among species. In this work, a methodology for the reconstruction and comparison of GCNs is presented. This approach was applied using gene expression data that were obtained from immunity experiments in Arabidopsis thaliana, rice, soybean, tomato and cassava. After the evaluation of diverse similarity metrics for the GCN reconstruction, we recommended the mutual information coefficient measurement and a clustering coefficient-based method for similarity threshold selection. To compare GCNs, we proposed a multivariate approach based on the Principal Component Analysis (PCA. Branches of plant immunity that were exemplified by each experiment were analyzed in conjunction with the PCA results, suggesting both the robustness and the dynamic nature of the cellular responses. The dynamic of molecular plant responses produced networks with different characteristics that are differentiable using our methodology. The comparison of GCNs from plant pathosystems, showed that in response to similar pathogens plants could activate conserved signaling pathways. The results confirmed that the closeness of GCNs projected on the principal component space is an indicative of similarity among GCNs. This also can be used to understand global patterns of events triggered during plant immune responses.

  6. Construction and comparison of gene co-expression networks shows complex plant immune responses.

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    Leal, Luis Guillermo; López, Camilo; López-Kleine, Liliana

    2014-01-01

    Gene co-expression networks (GCNs) are graphic representations that depict the coordinated transcription of genes in response to certain stimuli. GCNs provide functional annotations of genes whose function is unknown and are further used in studies of translational functional genomics among species. In this work, a methodology for the reconstruction and comparison of GCNs is presented. This approach was applied using gene expression data that were obtained from immunity experiments in Arabidopsis thaliana, rice, soybean, tomato and cassava. After the evaluation of diverse similarity metrics for the GCN reconstruction, we recommended the mutual information coefficient measurement and a clustering coefficient-based method for similarity threshold selection. To compare GCNs, we proposed a multivariate approach based on the Principal Component Analysis (PCA). Branches of plant immunity that were exemplified by each experiment were analyzed in conjunction with the PCA results, suggesting both the robustness and the dynamic nature of the cellular responses. The dynamic of molecular plant responses produced networks with different characteristics that are differentiable using our methodology. The comparison of GCNs from plant pathosystems, showed that in response to similar pathogens plants could activate conserved signaling pathways. The results confirmed that the closeness of GCNs projected on the principal component space is an indicative of similarity among GCNs. This also can be used to understand global patterns of events triggered during plant immune responses.

  7. Threshold selection in gene co-expression networks using spectral graph theory techniques.

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    Perkins, Andy D; Langston, Michael A

    2009-10-08

    Gene co-expression networks are often constructed by computing some measure of similarity between expression levels of gene transcripts and subsequently applying a high-pass filter to remove all but the most likely biologically-significant relationships. The selection of this expression threshold necessarily has a significant effect on any conclusions derived from the resulting network. Many approaches have been taken to choose an appropriate threshold, among them computing levels of statistical significance, accepting only the top one percent of relationships, and selecting an arbitrary expression cutoff. We apply spectral graph theory methods to develop a systematic method for threshold selection. Eigenvalues and eigenvectors are computed for a transformation of the adjacency matrix of the network constructed at various threshold values. From these, we use a basic spectral clustering method to examine the set of gene-gene relationships and select a threshold dependent upon the community structure of the data. This approach is applied to two well-studied microarray data sets from Homo sapiens and Saccharomyces cerevisiae. This method presents a systematic, data-based alternative to using more artificial cutoff values and results in a more conservative approach to threshold selection than some other popular techniques such as retaining only statistically-significant relationships or setting a cutoff to include a percentage of the highest correlations.

  8. Co-expression Network Approach Reveals Functional Similarities among Diseases Affecting Human Skeletal Muscle

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    Kavitha Mukund

    2017-12-01

    Full Text Available Diseases affecting skeletal muscle exhibit considerable heterogeneity in intensity, etiology, phenotypic manifestation and gene expression. Systems biology approaches using network theory, allows for a holistic understanding of functional similarities amongst diseases. Here we propose a co-expression based, network theoretic approach to extract functional similarities from 20 heterogeneous diseases comprising of dystrophinopathies, inflammatory myopathies, neuromuscular, and muscle metabolic diseases. Utilizing this framework we identified seven closely associated disease clusters with 20 disease pairs exhibiting significant correlation (p < 0.05. Mapping the diseases onto a human protein-protein interaction network enabled the inference of a common program of regulation underlying more than half the muscle diseases considered here and referred to as the “protein signature.” Enrichment analysis of 17 protein modules identified as part of this signature revealed a statistically non-random dysregulation of muscle bioenergetic pathways and calcium homeostasis. Further, analysis of mechanistic similarities of less explored significant disease associations [such as between amyotrophic lateral sclerosis (ALS and cerebral palsy (CP] using a proposed “functional module” framework revealed adaptation of the calcium signaling machinery. Integrating drug-gene information into the quantitative framework highlighted the presence of therapeutic opportunities through drug repurposing for diseases affecting the skeletal muscle.

  9. Different substrate regimes determine transcriptional profiles and gene co-expression in Methanosarcina barkeri (DSM 800).

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    Lin, Qiang; Fang, Xiaoyu; Ho, Adrian; Li, Jiaying; Yan, Xuefeng; Tu, Bo; Li, Chaonan; Li, Jiabao; Yao, Minjie; Li, Xiangzhen

    2017-08-21

    Methanosarcina barkeri (DSM 800) is a metabolically versatile methanogen and shows distinct metabolic status under different substrate regimes. However, the mechanisms underlying distinct transcriptional profiles under different substrate regimes remain elusive. In this study, based on transcriptional analysis, the growth performances and gene expressions of M. barkeri fed on acetate, H2 + CO2, and methanol, respectively, were investigated. M. barkeri showed higher growth performances under methanol, followed by H2 + CO2 and acetate, which corresponded well with the variations of gene expressions. The α diversity (evenness) of gene expressions was highest under the acetate regime, followed by H2 + CO2 and methanol, and significantly and negatively correlated with growth performances. The gene co-expression analysis showed that "Energy production and conversion," "Coenzyme transport and metabolism," and "Translation, ribosomal structure, and biogenesis" showed deterministic cooperation patterns of intra- and inter-functional classes. However, "Posttranslational modification, protein turnover, chaperones" showed exclusion with other functional classes. The gene expressions and especially the relationships among them potentially drove the shifts of metabolic status under different substrate regimes. Consequently, this study revealed the diversity-related ecological strategies that a high α diversity probably provided more fitness and tolerance under natural environments and oppositely a low α diversity strengthened some specific physiological functions, as well as the co-responses of gene expressions to different substrate regimes.

  10. Interactions of Na+,K+-ATPase and co-expressed delta-opioid receptor.

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    Deng, Haiping; Yang, Zhijie; Li, Yuting; Bao, Guobin; Friedrich, Thomas; Gu, Quanbao; Shen, Xueyong; Schwarz, Wolfgang

    2009-11-01

    To investigate interference of delta-opioid receptor with the Na(+),K(+)-ATPase in a simple model system, we used the Xenopus oocytes as an expression system. Our results indicate that expression of the delta-opioid receptor (DOR) results in reduction of endogenous sodium-pump activity. Stimulation of DOR by the DOR agonist [(D)-Pen(2,5)]-enkephalin (DPDPE) had no pronounced additional effect on pump activity. Qualitatively similar results were obtained in experiments with a variety of co-expressed exogenous sodium pumps. We suggest that reduced pump activity with DOR expression is brought about by an interaction of the pump with DOR. Direct interaction is also supported by co-immunoprecipitation, not only in the Xenopus oocytes but also in rat hippocampal neurons. The interaction may be responsible for altered agonist sensitivity of DOR; activation of the sodium pump led to an increase of the K(m) value for DOR activation by DPDPE from about 0.17 to 0.27muM. In conclusion, pump activity not only affects neural activity directly but our results also suggest that pump activity is affected through functional interaction with DOR that will modulate pain sensation.

  11. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

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    Choi Yoo

    2012-10-01

    Full Text Available Abstract Background In nature, mussel adhesive proteins (MAPs show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. Results A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Conclusion Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate

  12. Assisted protein folding at low temperature: evolutionary adaptation of the Antarctic fish chaperonin CCT and its client proteins

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    Cuellar, Jorge; Yébenes, Hugo; Parker, Sandra K.; Carranza, Gerardo; Serna, Marina; Valpuesta, José María; Zabala, Juan Carlos; Detrich, H. William

    2014-01-01

    ABSTRACT Eukaryotic ectotherms of the Southern Ocean face energetic challenges to protein folding assisted by the cytosolic chaperonin CCT. We hypothesize that CCT and its client proteins (CPs) have co-evolved molecular adaptations that facilitate CCT–CP interaction and the ATP-driven folding cycle at low temperature. To test this hypothesis, we compared the functional and structural properties of CCT–CP systems from testis tissues of an Antarctic fish, Gobionotothen gibberifrons (Lönnberg) (habitat/body T = −1.9 to +2°C), and of the cow (body T = 37°C). We examined the temperature dependence of the binding of denatured CPs (β-actin, β-tubulin) by fish and bovine CCTs, both in homologous and heterologous combinations and at temperatures between −4°C and 20°C, in a buffer conducive to binding of the denatured CP to the open conformation of CCT. In homologous combination, the percentage of G. gibberifrons CCT bound to CP declined linearly with increasing temperature, whereas the converse was true for bovine CCT. Binding of CCT to heterologous CPs was low, irrespective of temperature. When reactions were supplemented with ATP, G. gibberifrons CCT catalyzed the folding and release of actin at 2°C. The ATPase activity of apo-CCT from G. gibberifrons at 4°C was ∼2.5-fold greater than that of apo-bovine CCT, whereas equivalent activities were observed at 20°C. Based on these results, we conclude that the catalytic folding cycle of CCT from Antarctic fishes is partially compensated at their habitat temperature, probably by means of enhanced CP-binding affinity and increased flexibility of the CCT subunits. PMID:24659247

  13. Molecular diagnostic tools for detection and differentiation of phytoplasmas based on chaperonin-60 reveal differences in host plant infection patterns.

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    Tim J Dumonceaux

    Full Text Available Phytoplasmas ('Candidatus Phytoplasma' spp. are insect-vectored bacteria that infect a wide variety of plants, including many agriculturally important species. The infections can cause devastating yield losses by inducing morphological changes that dramatically alter inflorescence development. Detection of phytoplasma infection typically utilizes sequences located within the 16S-23S rRNA-encoding locus, and these sequences are necessary for strain identification by currently accepted standards for phytoplasma classification. However, these methods can generate PCR products >1400 bp that are less divergent in sequence than protein-encoding genes, limiting strain resolution in certain cases. We describe a method for accessing the chaperonin-60 (cpn60 gene sequence from a diverse array of 'Ca.Phytoplasma' spp. Two degenerate primer sets were designed based on the known sequence diversity of cpn60 from 'Ca.Phytoplasma' spp. and used to amplify cpn60 gene fragments from various reference samples and infected plant tissues. Forty three cpn60 sequences were thereby determined. The cpn60 PCR-gel electrophoresis method was highly sensitive compared to 16S-23S-targeted PCR-gel electrophoresis. The topology of a phylogenetic tree generated using cpn60 sequences was congruent with that reported for 16S rRNA-encoding genes. The cpn60 sequences were used to design a hybridization array using oligonucleotide-coupled fluorescent microspheres, providing rapid diagnosis and typing of phytoplasma infections. The oligonucleotide-coupled fluorescent microsphere assay revealed samples that were infected simultaneously with two subtypes of phytoplasma. These tools were applied to show that two host plants, Brassica napus and Camelina sativa, displayed different phytoplasma infection patterns.

  14. A yeast's eye view of mammalian reproduction: cross-species gene co-expression in meiotic prophase

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    Li Yunfei

    2010-09-01

    Full Text Available Abstract Background Meiotic prophase is a critical stage in sexual reproduction. Aberrant chromosome recombination during this stage is a leading cause of human miscarriages and birth defects. However, due to the experimental intractability of mammalian gonads, only a very limited number of meiotic genes have been characterized. Here we aim to identify novel meiotic genes important in human reproduction through computational mining of cross-species and cross-sex time-series expression data from budding yeast, mouse postnatal testis, mouse embryonic ovary, and human fetal ovary. Results Orthologous gene pairs were ranked by order statistics according to their co-expression profiles across species, allowing us to infer conserved meiotic genes despite obvious differences in cellular synchronicity and composition in organisms. We demonstrated that conserved co-expression networks could successfully recover known meiotic genes, including homologous recombination genes, chromatin cohesion genes, and genes regulating meiotic entry. We also showed that conserved co-expression pairs exhibit functional connections, as evidenced by the annotation similarity in Gene Ontology and overlap with physical interactions. More importantly, we predicted six new meiotic genes through their co-expression linkages with known meiotic genes, and subsequently used the genetically more amenable yeast system for experimental validation. The deletion mutants of all six genes showed sporulation defects, equivalent to a 100% validation rate. Conclusions We identified evolutionarily conserved gene modules in meiotic prophase by integrating cross-species and cross-sex expression profiles from budding yeast, mouse, and human. Our co-expression linkage analyses confirmed known meiotic genes and identified several novel genes that might be critical players in meiosis in multiple species. These results demonstrate that our approach is highly efficient to discover evolutionarily

  15. Discovering dysfunction of multiple microRNAs cooperation in disease by a conserved microRNA co-expression network.

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    Yun Xiao

    Full Text Available MicroRNAs, a new class of key regulators of gene expression, have been shown to be involved in diverse biological processes and linked to many human diseases. To elucidate miRNA function from a global perspective, we constructed a conserved miRNA co-expression network by integrating multiple human and mouse miRNA expression data. We found that these conserved co-expressed miRNA pairs tend to reside in close genomic proximity, belong to common families, share common transcription factors, and regulate common biological processes by targeting common components of those processes based on miRNA targets and miRNA knockout/transfection expression data, suggesting their strong functional associations. We also identified several co-expressed miRNA sub-networks. Our analysis reveals that many miRNAs in the same sub-network are associated with the same diseases. By mapping known disease miRNAs to the network, we identified three cancer-related miRNA sub-networks. Functional analyses based on targets and miRNA knockout/transfection data consistently show that these sub-networks are significantly involved in cancer-related biological processes, such as apoptosis and cell cycle. Our results imply that multiple co-expressed miRNAs can cooperatively regulate a given biological process by targeting common components of that process, and the pathogenesis of disease may be associated with the abnormality of multiple functionally cooperative miRNAs rather than individual miRNAs. In addition, many of these co-expression relationships provide strong evidence for the involvement of new miRNAs in important biological processes, such as apoptosis, differentiation and cell cycle, indicating their potential disease links.

  16. Evolution of Daily Gene Co-expression Patterns from Algae to Plants

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    Pedro de los Reyes

    2017-07-01

    Full Text Available Daily rhythms play a key role in transcriptome regulation in plants and microalgae orchestrating responses that, among other processes, anticipate light transitions that are essential for their metabolism and development. The recent accumulation of genome-wide transcriptomic data generated under alternating light:dark periods from plants and microalgae has made possible integrative and comparative analysis that could contribute to shed light on the evolution of daily rhythms in the green lineage. In this work, RNA-seq and microarray data generated over 24 h periods in different light regimes from the eudicot Arabidopsis thaliana and the microalgae Chlamydomonas reinhardtii and Ostreococcus tauri have been integrated and analyzed using gene co-expression networks. This analysis revealed a reduction in the size of the daily rhythmic transcriptome from around 90% in Ostreococcus, being heavily influenced by light transitions, to around 40% in Arabidopsis, where a certain independence from light transitions can be observed. A novel Multiple Bidirectional Best Hit (MBBH algorithm was applied to associate single genes with a family of potential orthologues from evolutionary distant species. Gene duplication, amplification and divergence of rhythmic expression profiles seems to have played a central role in the evolution of gene families in the green lineage such as Pseudo Response Regulators (PRRs, CONSTANS-Likes (COLs, and DNA-binding with One Finger (DOFs. Gene clustering and functional enrichment have been used to identify groups of genes with similar rhythmic gene expression patterns. The comparison of gene clusters between species based on potential orthologous relationships has unveiled a low to moderate level of conservation of daily rhythmic expression patterns. However, a strikingly high conservation was found for the gene clusters exhibiting their highest and/or lowest expression value during the light transitions.

  17. Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane.

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    Ferreira, Savio Siqueira; Hotta, Carlos Takeshi; Poelking, Viviane Guzzo de Carli; Leite, Debora Chaves Coelho; Buckeridge, Marcos Silveira; Loureiro, Marcelo Ehlers; Barbosa, Marcio Henrique Pereira; Carneiro, Monalisa Sampaio; Souza, Glaucia Mendes

    2016-05-01

    Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production.

  18. Weighted Gene Co-expression Network Analysis of the Dioscin Rich Medicinal Plant Dioscorea nipponica

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    Wei Sun

    2017-06-01

    Full Text Available Dioscorea contains critically important species which can be used as staple foods or sources of bioactive substances, including Dioscorea nipponica, which has been used to develop highly successful drugs to treat cardiovascular disease. Its major active ingredients are thought to be sterol compounds such as diosgenin, which has been called “medicinal gold” because of its valuable properties. However, reliance on naturally growing plants as a production system limits the potential use of D. nipponica, raising interest in engineering metabolic pathways to enhance the production of secondary metabolites. However, the biosynthetic pathway of diosgenin is still poorly understood, and D. nipponica is poorly characterized at a molecular level, hindering in-depth investigation. In the present work, the RNAs from five organs and seven methyl jasmonate treated D. nipponica rhizomes were sequenced using the Illumina high-throughput sequencing platform, yielding 52 gigabases of data, which were pooled and assembled into a reference transcriptome. Four hundred and eighty two genes were found to be highly expressed in the rhizomes, and these genes are mainly involved in stress response and transcriptional regulation. Based on their expression patterns, 36 genes were selected for further investigation as candidate genes involved in dioscin biosynthesis. Constructing co-expression networks based on significant changes in gene expression revealed 15 gene modules. Of these, four modules with properties correlating to dioscin regulation and biosynthesis, consisting of 4,665 genes in total, were selected for further functional investigation. These results improve our understanding of dioscin biosynthesis in this important medicinal plant and will help guide more intensive investigations.

  19. OsCpn60α1, encoding the plastid chaperonin 60α subunit, is essential for folding of rbcL.

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    Kim, Sung-Ryul; Yang, Jung-Il; An, Gynheung

    2013-05-01

    Chaperonins are involved in protein-folding. The rice genome encodes six plastid chaperonin subunits (Cpn60) - three α and three β. Our study showed that they were differentially expressed during normal plant development. Moreover, five were induced by heat stress (42°C) but not by cold (10°C). The oscpn60α1 mutant had a pale-green phenotype at the seedling stage and development ceased after the fourth leaf appeared. Transiently expressed OsCpn60α1:GFP fusion protein was localized to the chloroplast stroma. Immuno-blot analysis indicated that the level of Rubisco large subunit (rbcL) was severely reduced in the mutant while levels were unchanged for some imported proteins, e.g., stromal heat shock protein 70 (Hsp70) and chlorophyll a/b binding protein 1 (Lhcb1). This demonstrated that OsCpn60α1 is required for the folding of rbcL and that failure of that process is seedling-lethal.

  20. Identification of novel motif patterns to decipher the promoter architecture of co-expressed genes in Arabidopsis thaliana.

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    López, Yosvany; Patil, Ashwini; Nakai, Kenta

    2013-10-16

    The understanding of the mechanisms of transcriptional regulation remains a challenge for molecular biologists in the post-genome era. It is hypothesized that the regulatory regions of genes expressed in the same tissue or cell type share a similar structure. Though several studies have analyzed the promoters of genes expressed in specific metazoan tissues or cells, little research has been done in plants. Hence finding specific patterns of motifs to explain the promoter architecture of co-expressed genes in plants could shed light on their transcription mechanism. We identified novel patterns of sets of motifs in promoters of genes co-expressed in four different plant structures (PSs) and in the entire plant in Arabidopsis thaliana. Sets of genes expressed in four PSs (flower, seed, root, shoot) and housekeeping genes expressed in the entire plant were taken from a database of co-expressed genes in A. thaliana. PS-specific motifs were predicted using three motif-discovery algorithms, 8 of which are novel, to the best of our knowledge. A support vector machine was trained using the average upstream distance of the identified motifs from the translation start site on both strands of binding sites. The correctly classified promoters per PS were used to construct specific patterns of sets of motifs to describe the promoter architecture of those co-expressed genes. The discovered PS-specific patterns were tested in the entire A. thaliana genome, correctly identifying 77.8%, 81.2%, 70.8% and 53.7% genes expressed in petal differentiation, synergid cells, root hair and trichome, as well as 88.4% housekeeping genes. We present five patterns of sets of motifs which describe the promoter architecture of co-expressed genes in five PSs with the ability to predict them from the entire A. thaliana genome. Based on these findings, we conclude that the positioning and orientation of transcription factor binding sites at specific distances from the translation start site is a

  1. Broad integration of expression maps and co-expression networks compassing novel gene functions in the brain.

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    Okamura-Oho, Yuko; Shimokawa, Kazuro; Nishimura, Masaomi; Takemoto, Satoko; Sato, Akira; Furuichi, Teiichi; Yokota, Hideo

    2014-11-10

    Using a recently invented technique for gene expression mapping in the whole-anatomy context, termed transcriptome tomography, we have generated a dataset of 36,000 maps of overall gene expression in the adult-mouse brain. Here, using an informatics approach, we identified a broad co-expression network that follows an inverse power law and is rich in functional interaction and gene-ontology terms. Our framework for the integrated analysis of expression maps and graphs of co-expression networks revealed that groups of combinatorially expressed genes, which regulate cell differentiation during development, were present in the adult brain and each of these groups was associated with a discrete cell types. These groups included non-coding genes of unknown function. We found that these genes specifically linked developmentally conserved groups in the network. A previously unrecognized robust expression pattern covering the whole brain was related to the molecular anatomy of key biological processes occurring in particular areas.

  2. Recursive Indirect-Paths Modularity (RIP-M) for Detecting Community Structure in RNA-Seq Co-expression Networks.

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    Rahmani, Bahareh; Zimmermann, Michael T; Grill, Diane E; Kennedy, Richard B; Oberg, Ann L; White, Bill C; Poland, Gregory A; McKinney, Brett A

    2016-01-01

    Clusters of genes in co-expression networks are commonly used as functional units for gene set enrichment detection and increasingly as features (attribute construction) for statistical inference and sample classification. One of the practical challenges of clustering for these purposes is to identify an optimal partition of the network where the individual clusters are neither too large, prohibiting interpretation, nor too small, precluding general inference. Newman Modularity is a spectral clustering algorithm that automatically finds the number of clusters, but for many biological networks the cluster sizes are suboptimal. In this work, we generalize Newman Modularity to incorporate information from indirect paths in RNA-Seq co-expression networks. We implement a merge-and-split algorithm that allows the user to constrain the range of cluster sizes: large enough to capture genes in relevant pathways, yet small enough to resolve distinct functions. We investigate the properties of our recursive indirect-pathways modularity (RIP-M) and compare it with other clustering methods using simulated co-expression networks and RNA-seq data from an influenza vaccine response study. RIP-M had higher cluster assignment accuracy than Newman Modularity for finding clusters in simulated co-expression networks for all scenarios, and RIP-M had comparable accuracy to Weighted Gene Correlation Network Analysis (WGCNA). RIP-M was more accurate than WGCNA for modest hard thresholds and comparable for high, while WGCNA was slightly more accurate for soft thresholds. In the vaccine study data, RIP-M and WGCNA enriched for a comparable number of immunologically relevant pathways.

  3. Co-expression of two heterologous lactate dehydrogenases genes in Kluyveromyces marxianus for l-lactic acid production.

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    Lee, Jae Won; In, Jung Hoon; Park, Joon-Bum; Shin, Jonghyeok; Park, Jin Hwan; Sung, Bong Hyun; Sohn, Jung-Hoon; Seo, Jin-Ho; Park, Jin-Byoung; Kim, Soo Rin; Kweon, Dae-Hyuk

    2017-01-10

    Lactic acid (LA) is a versatile compound used in the food, pharmaceutical, textile, leather, and chemical industries. Biological production of LA is possible by yeast strains expressing a bacterial gene encoding l-lactate dehydrogenase (LDH). Kluyveromyces marxianus is an emerging non-conventional yeast with various phenotypes of industrial interest. However, it has not been extensively studied for LA production. In this study, K. marxianus was engineered to express and co-express various heterologous LDH enzymes that were reported to have different pH optimums. Specifically, three LDH enzymes originating from Staphylococcus epidermidis (SeLDH; optimal at pH 5.6), Lactobacillus acidophilus (LaLDH; optimal at pH 5.3), and Bos taurus (BtLDH; optimal at pH 9.8) were functionally expressed individually and in combination in K. marxianus, and the resulting strains were compared in terms of LA production. A strain co-expressing SeLDH and LaLDH (KM5 La+SeLDH) produced 16.0g/L LA, whereas the strains expressing those enzymes individually produced only 8.4 and 6.8g/L, respectively. This co-expressing strain produced 24.0g/L LA with a yield of 0.48g/g glucose in the presence of CaCO3. Our results suggest that co-expression of LDH enzymes with different pH optimums provides sufficient LDH activity under dynamic intracellular pH conditions, leading to enhanced production of LA compared to individual expression of the LDH enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. ARNetMiT R Package: association rules based gene co-expression networks of miRNA targets.

    Science.gov (United States)

    Özgür Cingiz, M; Biricik, G; Diri, B

    2017-03-31

    miRNAs are key regulators that bind to target genes to suppress their gene expression level. The relations between miRNA-target genes enable users to derive co-expressed genes that may be involved in similar biological processes and functions in cells. We hypothesize that target genes of miRNAs are co-expressed, when they are regulated by multiple miRNAs. With the usage of these co-expressed genes, we can theoretically construct co-expression networks (GCNs) related to 152 diseases. In this study, we introduce ARNetMiT that utilize a hash based association rule algorithm in a novel way to infer the GCNs on miRNA-target genes data. We also present R package of ARNetMiT, which infers and visualizes GCNs of diseases that are selected by users. Our approach assumes miRNAs as transactions and target genes as their items. Support and confidence values are used to prune association rules on miRNA-target genes data to construct support based GCNs (sGCNs) along with support and confidence based GCNs (scGCNs). We use overlap analysis and the topological features for the performance analysis of GCNs. We also infer GCNs with popular GNI algorithms for comparison with the GCNs of ARNetMiT. Overlap analysis results show that ARNetMiT outperforms the compared GNI algorithms. We see that using high confidence values in scGCNs increase the ratio of the overlapped gene-gene interactions between the compared methods. According to the evaluation of the topological features of ARNetMiT based GCNs, the degrees of nodes have power-law distribution. The hub genes discovered by ARNetMiT based GCNs are consistent with the literature.

  5. Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C.

    Science.gov (United States)

    Su, Lingqia; Jiang, Qi; Yu, Lingang; Wu, Jing

    2017-02-08

    Our laboratory has reported a strategy for improving the extracellular production of recombinant proteins through co-expression with Thermobifida fusca cutinase, which increases membrane permeability via its phospholipid hydrolysis activity. However, the foam generated by the lysophospholipid product makes the fermentation process difficult to control in a fermentor. Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce sn1,2-diacylglycerides and organic phosphate, which do not induce foam formation. Therefore, co-expression with Bacillus cereus PLC was investigated as a method to improve the extracellular production of recombinant proteins. When B. cereus PLC was expressed in Escherichia coli without its signal peptide, 95.3% of the total PLC activity was detected in the culture supernatant. PLC expression enhanced membrane permeability without obvious cell lysis. Then, six test enzymes, three secretory and three cytosolic, were co-expressed with B. cereus PLC. The enhancement of extracellular production correlated strongly with the molecular mass of the test enzyme. Extracellular production of Streptomyces sp. FA1 xylanase (43 kDa), which had the lowest molecular mass among the secretory enzymes, was 4.0-fold that of its individual expression control. Extracellular production of glutamate decarboxylase (51 kDa), which had the lowest molecular mass among the cytosolic enzymes, reached 26.7 U/mL; 88.3% of the total activity produced. This strategy was effectively scaled up using a 3-L fermentor. No obvious foam was generated during this fermentation process. This is the first study to detail the enhanced extracellular production of recombinant proteins through co-expression with PLC. This new strategy, which is especially appropriate for lower molecular mass proteins, allows large-scale protein production in an easily controlled fermentation process.

  6. Reconstruction of an integrated genome-scale co-expression network reveals key modules involved in lung adenocarcinoma.

    Science.gov (United States)

    Bidkhori, Gholamreza; Narimani, Zahra; Hosseini Ashtiani, Saman; Moeini, Ali; Nowzari-Dalini, Abbas; Masoudi-Nejad, Ali

    2013-01-01

    Our goal of this study was to reconstruct a "genome-scale co-expression network" and find important modules in lung adenocarcinoma so that we could identify the genes involved in lung adenocarcinoma. We integrated gene mutation, GWAS, CGH, array-CGH and SNP array data in order to identify important genes and loci in genome-scale. Afterwards, on the basis of the identified genes a co-expression network was reconstructed from the co-expression data. The reconstructed network was named "genome-scale co-expression network". As the next step, 23 key modules were disclosed through clustering. In this study a number of genes have been identified for the first time to be implicated in lung adenocarcinoma by analyzing the modules. The genes EGFR, PIK3CA, TAF15, XIAP, VAPB, Appl1, Rab5a, ARF4, CLPTM1L, SP4, ZNF124, LPP, FOXP1, SOX18, MSX2, NFE2L2, SMARCC1, TRA2B, CBX3, PRPF6, ATP6V1C1, MYBBP1A, MACF1, GRM2, TBXA2R, PRKAR2A, PTK2, PGF and MYO10 are among the genes that belong to modules 1 and 22. All these genes, being implicated in at least one of the phenomena, namely cell survival, proliferation and metastasis, have an over-expression pattern similar to that of EGFR. In few modules, the genes such as CCNA2 (Cyclin A2), CCNB2 (Cyclin B2), CDK1, CDK5, CDC27, CDCA5, CDCA8, ASPM, BUB1, KIF15, KIF2C, NEK2, NUSAP1, PRC1, SMC4, SYCE2, TFDP1, CDC42 and ARHGEF9 are present that play a crucial role in cell cycle progression. In addition to the mentioned genes, there are some other genes (i.e. DLGAP5, BIRC5, PSMD2, Src, TTK, SENP2, PSMD2, DOK2, FUS and etc.) in the modules.

  7. Chaperonin containing T-complex polypeptide subunit eta (CCT-eta is a specific regulator of fibroblast motility and contractility.

    Directory of Open Access Journals (Sweden)

    Latha Satish

    2010-04-01

    Full Text Available Integumentary wounds in mammalian fetuses heal without scar; this scarless wound healing is intrinsic to fetal tissues and is notable for absence of the contraction seen in postnatal (adult wounds. The precise molecular signals determining the scarless phenotype remain unclear. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta is specifically reduced in healing fetal wounds in a rabbit model. In this study, we examine the role of CCT-eta in fibroblast motility and contractility, properties essential to wound healing and scar formation. We demonstrate that CCT-eta (but not CCT-beta is underexpressed in fetal fibroblasts compared to adult fibroblasts. An in vitro wound healing assay demonstrated that adult fibroblasts showed increased cell migration in response to epidermal growth factor (EGF and platelet derived growth factor (PDGF stimulation, whereas fetal fibroblasts were unresponsive. Downregulation of CCT-eta in adult fibroblasts with short inhibitory RNA (siRNA reduced cellular motility, both basal and growth factor-induced; in contrast, siRNA against CCT-beta had no such effect. Adult fibroblasts were more inherently contractile than fetal fibroblasts by cellular traction force microscopy; this contractility was increased by treatment with EGF and PDGF. CCT-eta siRNA inhibited the PDGF-induction of adult fibroblast contractility, whereas CCT-beta siRNA had no such effect. In each of these instances, the effect of downregulating CCT-eta was to modulate the behavior of adult fibroblasts so as to more closely approximate the characteristics of fetal fibroblasts. We next examined the effect of CCT-eta modulation on alpha-smooth muscle actin (alpha-SMA expression, a gene product well known to play a critical role in adult wound healing. Fetal fibroblasts were found to constitutively express less alpha-SMA than adult cells. Reduction of CCT-eta with siRNA had minimal effect on cellular

  8. Systematic comparison of co-expression of multiple recombinant thermophilic enzymes in Escherichia coli BL21(DE3).

    Science.gov (United States)

    Chen, Hui; Huang, Rui; Zhang, Y-H Percival

    2017-06-01

    The precise control of multiple heterologous enzyme expression levels in one Escherichia coli strain is important for cascade biocatalysis, metabolic engineering, synthetic biology, natural product synthesis, and studies of complexed proteins. We systematically investigated the co-expression of up to four thermophilic enzymes (i.e., α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), glucose 6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH)) in E. coli BL21(DE3) by adding T7 promoter or T7 terminator of each gene for multiple genes in tandem, changing gene alignment, and comparing one or two plasmid systems. It was found that the addition of T7 terminator after each gene was useful to decrease the influence of the upstream gene. The co-expression of the four enzymes in E. coli BL21(DE3) was demonstrated to generate two NADPH molecules from one glucose unit of maltodextrin, where NADPH was oxidized to convert xylose to xylitol. The best four-gene co-expression system was based on two plasmids (pET and pACYC) which harbored two genes. As a result, apparent enzymatic activities of the four enzymes were regulated to be at similar levels and the overall four-enzyme activity was the highest based on the formation of xylitol. This study provides useful information for the precise control of multi-enzyme-coordinated expression in E. coli BL21(DE3).

  9. Genes and co-expression modules common to drought and bacterial stress responses in Arabidopsis and rice.

    Science.gov (United States)

    Shaik, Rafi; Ramakrishna, Wusirika

    2013-01-01

    Plants are simultaneously exposed to multiple stresses resulting in enormous changes in the molecular landscape within the cell. Identification and characterization of the synergistic and antagonistic components of stress response mechanisms contributing to the cross talk between stresses is of high priority to explore and enhance multiple stress responses. To this end, we performed meta-analysis of drought (abiotic), bacterial (biotic) stress response in rice and Arabidopsis by analyzing a total of 386 microarray samples belonging to 20 microarray studies and identified approximately 3100 and 900 DEGs in rice and Arabidopsis, respectively. About 38.5% (1214) and 28.7% (272) DEGs were common to drought and bacterial stresses in rice and Arabidopsis, respectively. A majority of these common DEGs showed conserved expression status in both stresses. Gene ontology enrichment analysis clearly demarcated the response and regulation of various plant hormones and related biological processes. Fatty acid metabolism and biosynthesis of alkaloids were upregulated and, nitrogen metabolism and photosynthesis was downregulated in both stress conditions. WRKY transcription family genes were highly enriched in all upregulated gene sets while 'CO-like' TF family showed inverse relationship of expression between drought and bacterial stresses. Weighted gene co-expression network analysis divided DEG sets into multiple modules that show high co-expression and identified stress specific hub genes with high connectivity. Detection of consensus modules based on DEGs common to drought and bacterial stress revealed 9 and 4 modules in rice and Arabidopsis, respectively, with conserved and reversed co-expression patterns.

  10. Positive prognostic value of HER2-HER3 co-expression and p-mTOR in gastric cancer patients.

    Science.gov (United States)

    Cao, Guo-Dong; Chen, Ke; Chen, Bo; Xiong, Mao-Ming

    2017-12-12

    The HER2-HER3 heterodimer significantly decreases survival in breast cancer patients. However, the prognostic value of HER2-HER3 overexpression remains unknown in gastric cancer (GC). The expression levels of HER2, HER3, Akt, p-Akt, mTOR and p-mTOR were examined in specimens from 120 GC patients by immunohistochemistry and quantitative reverse transcription-PCR. The associations of HER proteins, PI3K/Akt/mTOR pathway-related proteins, clinicopathological features of GC, and overall survival (OS) were assessed. To comprehensively evaluate the prognostic values of pathway-related proteins, meta-analyses were conducted with STATA 11.0. HER2 overexpression was significantly associated with HER3 levels (P = 0.02). HER3 was highly expressed in gastric cancer tissues. High HER2 and HER3 levels were associated with elevated p-Akt and p-mTOR amounts (P HER2-HER3 co-expression was associated with high p-Akt and p-mTOR (P HER2-HER3 overexpression corresponded to gradually shortened 5-year OS (P HER2-HER3 co-expression may potentially enhance mTOR phosphorylation. HER2-HER3 co-expression and p-mTOR are both related to the prognosis of GC patients.

  11. The novel neuropeptide phoenixin is highly co-expressed with nesfatin-1 in the rat hypothalamus, an immunohistochemical study.

    Science.gov (United States)

    Pałasz, Artur; Rojczyk, Ewa; Bogus, Katarzyna; Worthington, John J; Wiaderkiewicz, Ryszard

    2015-04-10

    The hypothalamus regulates a number of autonomic functions essential for homeostasis; therefore, investigations concerning hypothalamic neuropeptides and their functions and distribution are of great importance in contemporary neuroscience. Recently, novel regulatory factors expressed in the hypothalamus have been discovered, of which nesfatin-1 and phoenixin (PNX), show intriguing similarities in their brain distributions. There are currently few studies characterizing PNX expression, so it is imperative to accurately trace its localization, with particular attention to the hypothalamic nuclei and nesfatin-1 co-expression. Using fluorescence and classical immunohistochemical stainings on adult rat brain, we visualized the potential co-expression of nesfatin-1 and PNX immunoreactive cells. We have demonstrated a distinct PNX-immunoreactivity in 21-32% of cells in the arcuate nucleus, paraventricular nucleus, ventromedial and lateral hypothalamus. Nesfatin-1 expression reached 45-68% of all neurons in the same sites, while co-expression was strikingly seen in the vast majority (70-86%) of PNX-immunoreactive neurons in the rat hypothalamic nuclei. Our results demonstrate for the first time, a wide distribution of PNX in the hypothalamus which could implicate a potential functional relationship with nesfatin-1, possibly in the regulation of the hypothalamic-pituitary-gonadal axis or other autonomic functions, which require further study. Copyright © 2015. Published by Elsevier Ireland Ltd.

  12. Mycobacterium tuberculosis Chaperonin 10 Is Secreted in the Macrophage Phagosome: Is Secretion Due to Dissociation and Adoption of a Partially Helical Structure at the Membrane?

    Science.gov (United States)

    Fossati, Gianluca; Izzo, Gaetano; Rizzi, Emanuele; Gancia, Emanuela; Modena, Daniela; Moras, Maria Luisa; Niccolai, Neri; Giannozzi, Elena; Spiga, Ottavia; Bono, Letizia; Marone, Piero; Leone, Eugenio; Mangili, Francesca; Harding, Stephen; Errington, Neil; Walters, Christopher; Henderson, Brian; Roberts, Michael M.; Coates, Anthony R. M.; Casetta, Bruno; Mascagni, Paolo

    2003-01-01

    To confirm that Mycobacterium tuberculosis chaperonin 10 (Cpn10) is secreted outside the live bacillus, infected macrophages were examined by electron microscopy. This revealed that the mycobacterial protein accumulates both in the wall of the bacterium and in the matrix of the phagosomes in which ingested mycobacteria survive within infected macrophages. To understand the structural implications underlying this secretion, a structural study of M. tuberculosis Cpn10 was performed under conditions that are generally believed to mimic the membrane environment. It was found that in buffer-organic solvent mixtures, the mycobacterial protein forms two main species, namely, a partially helical monomer that prevails in dilute solutions at room temperature and a dimer that folds into a β-sheet-dominated structure and prevails in either concentrated protein solutions at room temperature or in dilute solutions at low temperature. A partially helical monomer was also found and was completely associated with negatively charged detergents in a micelle-bound state. Remarkably, zwitterionic lipids had no effect on the protein structure. By using N- and C-truncated forms of the protein, the C- and N-terminal sequences were identified as possessing an amphiphilic helical character and as selectively associating with acidic detergent micelles. When the study was extended to other chaperonins, it was found that human Cpn10 is also monomeric and partially helical in dilute organic solvent-buffer mixtures. In contrast, Escherichia coli Cpn10 is mostly dimeric and predominately β-sheet in both dilute and concentrated solutions. Interestingly, human Cpn10 also crosses biological membranes, whereas the E. coli homologue is strictly cytosolic. These results suggest that dissociation to partially helical monomers and interaction with acidic lipids may be two important steps in the mechanism of secretion of M. tuberculosis Cpn10 to the external environment. PMID:12837802

  13. Identification of a Novel BBS Gene (BBS12) Highlights the Major Role of a Vertebrate-Specific Branch of Chaperonin-Related Proteins in Bardet-Biedl Syndrome

    Science.gov (United States)

    Stoetzel, Corinne; Muller, Jean; Laurier, Virginie; Davis, Erica E.; Zaghloul, Norann A.; Vicaire, Serge; Jacquelin, Cécile; Plewniak, Frédéric; Leitch, Carmen C.; Sarda, Pierre; Hamel, Christian; de Ravel, Thomy J. L.; Lewis, Richard Alan; Friederich, Evelyne; Thibault, Christelle; Danse, Jean-Marc; Verloes, Alain; Bonneau, Dominique; Katsanis, Nicholas; Poch, Olivier; Mandel, Jean-Louis; Dollfus, Hélène

    2007-01-01

    Bardet-Biedl syndrome (BBS) is primarily an autosomal recessive ciliopathy characterized by progressive retinal degeneration, obesity, cognitive impairment, polydactyly, and kidney anomalies. The disorder is genetically heterogeneous, with 11 BBS genes identified to date, which account for ∼70% of affected families. We have combined single-nucleotide–polymorphism array homozygosity mapping with in silico analysis to identify a new BBS gene, BBS12. Patients from two Gypsy families were homozygous and haploidentical in a 6-Mb region of chromosome 4q27. FLJ35630 was selected as a candidate gene, because it was predicted to encode a protein with similarity to members of the type II chaperonin superfamily, which includes BBS6 and BBS10. We found pathogenic mutations in both Gypsy families, as well as in 14 other families of various ethnic backgrounds, indicating that BBS12 accounts for ∼5% of all BBS cases. BBS12 is vertebrate specific and, together with BBS6 and BBS10, defines a novel branch of the type II chaperonin superfamily. These three genes are characterized by unusually rapid evolution and are likely to perform ciliary functions specific to vertebrates that are important in the pathophysiology of the syndrome, and together they account for about one-third of the total BBS mutational load. Consistent with this notion, suppression of each family member in zebrafish yielded gastrulation-movement defects characteristic of other BBS morphants, whereas simultaneous suppression of all three members resulted in severely affected embryos, possibly hinting at partial functional redundancy within this protein family. PMID:17160889

  14. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    Directory of Open Access Journals (Sweden)

    Pandey Sona

    2010-11-01

    Full Text Available Abstract Background Cytochrome P450 monooxygenases (P450s catalyze oxidation of various substrates using oxygen and NAD(PH. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship. Results We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an

  15. Using Co-Expression Analysis and Stress-Based Screens to Uncover Arabidopsis Peroxisomal Proteins Involved in Drought Response.

    Directory of Open Access Journals (Sweden)

    Jiying Li

    Full Text Available Peroxisomes are essential organelles that house a wide array of metabolic reactions important for plant growth and development. However, our knowledge regarding the role of peroxisomal proteins in various biological processes, including plant stress response, is still incomplete. Recent proteomic studies of plant peroxisomes significantly increased the number of known peroxisomal proteins and greatly facilitated the study of peroxisomes at the systems level. The objectives of this study were to determine whether genes that encode peroxisomal proteins with related functions are co-expressed in Arabidopsis and identify peroxisomal proteins involved in stress response using in silico analysis and mutant screens. Using microarray data from online databases, we performed hierarchical clustering analysis to generate a comprehensive view of transcript level changes for Arabidopsis peroxisomal genes during development and under abiotic and biotic stress conditions. Many genes involved in the same metabolic pathways exhibited co-expression, some genes known to be involved in stress response are regulated by the corresponding stress conditions, and function of some peroxisomal proteins could be predicted based on their co-expression pattern. Since drought caused expression changes to the highest number of genes that encode peroxisomal proteins, we subjected a subset of Arabidopsis peroxisomal mutants to a drought stress assay. Mutants of the LON2 protease and the photorespiratory enzyme hydroxypyruvate reductase 1 (HPR1 showed enhanced susceptibility to drought, suggesting the involvement of peroxisomal quality control and photorespiration in drought resistance. Our study provided a global view of how genes that encode peroxisomal proteins respond to developmental and environmental cues and began to reveal additional peroxisomal proteins involved in stress response, thus opening up new avenues to investigate the role of peroxisomes in plant adaptation to

  16. Genes and co-expression modules common to drought and bacterial stress responses in Arabidopsis and rice.

    Directory of Open Access Journals (Sweden)

    Rafi Shaik

    Full Text Available Plants are simultaneously exposed to multiple stresses resulting in enormous changes in the molecular landscape within the cell. Identification and characterization of the synergistic and antagonistic components of stress response mechanisms contributing to the cross talk between stresses is of high priority to explore and enhance multiple stress responses. To this end, we performed meta-analysis of drought (abiotic, bacterial (biotic stress response in rice and Arabidopsis by analyzing a total of 386 microarray samples belonging to 20 microarray studies and identified approximately 3100 and 900 DEGs in rice and Arabidopsis, respectively. About 38.5% (1214 and 28.7% (272 DEGs were common to drought and bacterial stresses in rice and Arabidopsis, respectively. A majority of these common DEGs showed conserved expression status in both stresses. Gene ontology enrichment analysis clearly demarcated the response and regulation of various plant hormones and related biological processes. Fatty acid metabolism and biosynthesis of alkaloids were upregulated and, nitrogen metabolism and photosynthesis was downregulated in both stress conditions. WRKY transcription family genes were highly enriched in all upregulated gene sets while 'CO-like' TF family showed inverse relationship of expression between drought and bacterial stresses. Weighted gene co-expression network analysis divided DEG sets into multiple modules that show high co-expression and identified stress specific hub genes with high connectivity. Detection of consensus modules based on DEGs common to drought and bacterial stress revealed 9 and 4 modules in rice and Arabidopsis, respectively, with conserved and reversed co-expression patterns.

  17. Co-expression of CD147 and GLUT-1 indicates radiation resistance and poor prognosis in cervical squamous cell carcinoma.

    Science.gov (United States)

    Huang, Xin-Qiong; Chen, Xiang; Xie, Xiao-Xue; Zhou, Qin; Li, Kai; Li, Shan; Shen, Liang-Fang; Su, Juan

    2014-01-01

    The aim of this study was to investigate the association of CD147 and GLUT-1, which play important roles in glycolysis in response to radiotherapy and clinical outcomes in patients with locally advanced cervical squamous cell carcinoma (LACSCC). The records of 132 female patients who received primary radiation therapy to treat LACSCC at FIGO stages IB-IVA were retrospectively reviewed. Forty-seven patients with PFS (progression-free survival) of less than 36 months were regarded as radiation-resistant. Eighty-five patients with PFS longer than 36 months were regarded as radiation-sensitive. Using pretreatment paraffin-embedded tissues, we evaluated CD147 and GLUT-1 expression by immunohistochemistry. Overexpression of CD147, GLUT-1, and CD147 and GLUT-1 combined were 44.7%, 52.9% and 36.5%, respectively, in the radiation-sensitive group, and 91.5%, 89.4% and 83.0%, respectively, in the radiation-resistant group. The 5-year progress free survival (PFS) rates in the CD147-low, CD147-high, GLUT-1-low, GLUT-1-high, CD147- and/or GLUT-1-low and CD147- and GLUT-1- dual high expression groups were 66.79%, 87.10%, 52.78%, 85.82%, 55.94%, 82.90% and 50.82%, respectively. CD147 and GLUT-1 co-expression, FIGO stage and tumor diameter were independent poor prognostic factors for patients with LACSCC in multivariate Cox regression analysis. Patients with high expression of CD147 alone, GLUT-1 alone or co-expression of CD147 and GLUT-1 showed greater resistance to radiotherapy and a shorter PFS than those with low expression. In particular, co-expression of CD147 and GLUT-1 can be considered as a negative independent prognostic factor.

  18. The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter

    DEFF Research Database (Denmark)

    Darbani Shirvanehdeh, Behrooz; Motawie, Mohammed Saddik; Olsen, Carl Erik

    2016-01-01

    for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated...... experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O...

  19. Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase

    Science.gov (United States)

    2012-01-01

    Background Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H2) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H2 production yield under light conditions. Results Recombinant E. coli BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H2 in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m2·s)). We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H2 production (~1.3-fold more) with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m2·s) led to an increase (~1.8-fold) in H2 production from 287.3 to 525.7 mL H2/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H2 achieved in this study was ~3.4%. Conclusion Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H2 production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3) in a light intensity-dependent manner. These results demonstrate that E. coli can be applied as

  20. Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase

    Directory of Open Access Journals (Sweden)

    Kim Jaoon YH

    2012-01-01

    Full Text Available Abstract Background Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H2 production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H2 production yield under light conditions. Results Recombinant E. coli BL21(DE3 co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H2 in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m2·s. We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H2 production (~1.3-fold more with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m2·s led to an increase (~1.8-fold in H2 production from 287.3 to 525.7 mL H2/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H2 achieved in this study was ~3.4%. Conclusion Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H2 production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3 in a light intensity-dependent manner. These results demonstrate that E. coli

  1. Reconstruction of an integrated genome-scale co-expression network reveals key modules involved in lung adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Gholamreza Bidkhori

    Full Text Available Our goal of this study was to reconstruct a "genome-scale co-expression network" and find important modules in lung adenocarcinoma so that we could identify the genes involved in lung adenocarcinoma. We integrated gene mutation, GWAS, CGH, array-CGH and SNP array data in order to identify important genes and loci in genome-scale. Afterwards, on the basis of the identified genes a co-expression network was reconstructed from the co-expression data. The reconstructed network was named "genome-scale co-expression network". As the next step, 23 key modules were disclosed through clustering. In this study a number of genes have been identified for the first time to be implicated in lung adenocarcinoma by analyzing the modules. The genes EGFR, PIK3CA, TAF15, XIAP, VAPB, Appl1, Rab5a, ARF4, CLPTM1L, SP4, ZNF124, LPP, FOXP1, SOX18, MSX2, NFE2L2, SMARCC1, TRA2B, CBX3, PRPF6, ATP6V1C1, MYBBP1A, MACF1, GRM2, TBXA2R, PRKAR2A, PTK2, PGF and MYO10 are among the genes that belong to modules 1 and 22. All these genes, being implicated in at least one of the phenomena, namely cell survival, proliferation and metastasis, have an over-expression pattern similar to that of EGFR. In few modules, the genes such as CCNA2 (Cyclin A2, CCNB2 (Cyclin B2, CDK1, CDK5, CDC27, CDCA5, CDCA8, ASPM, BUB1, KIF15, KIF2C, NEK2, NUSAP1, PRC1, SMC4, SYCE2, TFDP1, CDC42 and ARHGEF9 are present that play a crucial role in cell cycle progression. In addition to the mentioned genes, there are some other genes (i.e. DLGAP5, BIRC5, PSMD2, Src, TTK, SENP2, PSMD2, DOK2, FUS and etc. in the modules.

  2. Gene co-expression network analysis in Rhodobacter capsulatus and application to comparative expression analysis of Rhodobacter sphaeroides

    Energy Technology Data Exchange (ETDEWEB)

    Pena-Castillo, Lourdes; Mercer, Ryan; Gurinovich, Anastasia; Callister, Stephen J.; Wright, Aaron T.; Westbye, Alexander; Beatty, J. T.; Lang, Andrew S.

    2014-08-28

    The genus Rhodobacter contains purple nonsulfur bacteria found mostly in freshwater environments. Representative strains of two Rhodobacter species, R. capsulatus and R. sphaeroides, have had their genomes fully sequenced and both have been the subject of transcriptional profiling studies. Gene co-expression networks can be used to identify modules of genes with similar expression profiles. Functional analysis of gene modules can then associate co-expressed genes with biological pathways, and network statistics can determine the degree of module preservation in related networks. In this paper, we constructed an R. capsulatus gene co-expression network, performed functional analysis of identified gene modules, and investigated preservation of these modules in R. capsulatus proteomics data and in R. sphaeroides transcriptomics data. Results: The analysis identified 40 gene co-expression modules in R. capsulatus. Investigation of the module gene contents and expression profiles revealed patterns that were validated based on previous studies supporting the biological relevance of these modules. We identified two R. capsulatus gene modules preserved in the protein abundance data. We also identified several gene modules preserved between both Rhodobacter species, which indicate that these cellular processes are conserved between the species and are candidates for functional information transfer between species. Many gene modules were non-preserved, providing insight into processes that differentiate the two species. In addition, using Local Network Similarity (LNS), a recently proposed metric for expression divergence, we assessed the expression conservation of between-species pairs of orthologs, and within-species gene-protein expression profiles. Conclusions: Our analyses provide new sources of information for functional annotation in R. capsulatus because uncharacterized genes in modules are now connected with groups of genes that constitute a joint functional

  3. Genetic architecture of wood properties based on association analysis and co-expression networks in white spruce.

    Science.gov (United States)

    Lamara, Mebarek; Raherison, Elie; Lenz, Patrick; Beaulieu, Jean; Bousquet, Jean; MacKay, John

    2016-04-01

    Association studies are widely utilized to analyze complex traits but their ability to disclose genetic architectures is often limited by statistical constraints, and functional insights are usually minimal in nonmodel organisms like forest trees. We developed an approach to integrate association mapping results with co-expression networks. We tested single nucleotide polymorphisms (SNPs) in 2652 candidate genes for statistical associations with wood density, stiffness, microfibril angle and ring width in a population of 1694 white spruce trees (Picea glauca). Associations mapping identified 229-292 genes per wood trait using a statistical significance level of P wood associated genes and several known MYB and NAC regulators were identified as network hubs. The network revealed a link between the gene PgNAC8, wood stiffness and microfibril angle, as well as considerable within-season variation for both genetic control of wood traits and gene expression. Trait associations were distributed throughout the network suggesting complex interactions and pleiotropic effects. Our findings indicate that integration of association mapping and co-expression networks enhances our understanding of complex wood traits. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  4. Integration of Metabolic Modeling with Gene Co-expression Reveals Transcriptionally Programmed Reactions Explaining Robustness in Mycobacterium tuberculosis

    Science.gov (United States)

    Puniya, Bhanwar Lal; Kulshreshtha, Deepika; Mittal, Inna; Mobeen, Ahmed; Ramachandran, Srinivasan

    2016-01-01

    Robustness of metabolic networks is accomplished by gene regulation, modularity, re-routing of metabolites and plasticity. Here, we probed robustness against perturbations of biochemical reactions of M. tuberculosis in the form of predicting compensatory trends. In order to investigate the transcriptional programming of genes associated with correlated fluxes, we integrated with gene co-expression network. Knock down of the reactions NADH2r and ATPS responsible for producing the hub metabolites, and Central carbon metabolism had the highest proportion of their associated genes under transcriptional co-expression with genes of their flux correlated reactions. Reciprocal gene expression correlations were observed among compensatory routes, fresh activation of alternative routes and in the multi-copy genes of Cysteine synthase and of Phosphate transporter. Knock down of 46 reactions caused the activation of Isocitrate lyase or Malate synthase or both reactions, which are central to the persistent state of M. tuberculosis. A total of 30 new freshly activated routes including Cytochrome c oxidase, Lactate dehydrogenase, and Glycine cleavage system were predicted, which could be responsible for switching into dormant or persistent state. Thus, our integrated approach of exploring transcriptional programming of flux correlated reactions has the potential to unravel features of system architecture conferring robustness. PMID:27000948

  5. Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

    Science.gov (United States)

    Wang, Rongzhi; Xiang, Shuangshuang; Feng, Youjun; Srinivas, Swaminath; Zhang, Yonghui; Lin, Mingshen; Wang, Shihua

    2013-01-01

    Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility, and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody. PMID:24224158

  6. Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

    Directory of Open Access Journals (Sweden)

    Rongzhi eWang

    2013-11-01

    Full Text Available Single-chain variable fragment (scFv is a class of engineered antibodies generated by the fusion of the heavy (VH and light chains (VL of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.

  7. Identification of candidate genes in Populus cell wall biosynthesis using text-mining, co-expression network and comparative genomics

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xiaohan [ORNL; Ye, Chuyu [ORNL; Bisaria, Anjali [ORNL; Tuskan, Gerald A [ORNL; Kalluri, Udaya C [ORNL

    2011-01-01

    Populus is an important bioenergy crop for bioethanol production. A greater understanding of cell wall biosynthesis processes is critical in reducing biomass recalcitrance, a major hindrance in efficient generation of ethanol from lignocellulosic biomass. Here, we report the identification of candidate cell wall biosynthesis genes through the development and application of a novel bioinformatics pipeline. As a first step, via text-mining of PubMed publications, we obtained 121 Arabidopsis genes that had the experimental evidences supporting their involvement in cell wall biosynthesis or remodeling. The 121 genes were then used as bait genes to query an Arabidopsis co-expression database and additional genes were identified as neighbors of the bait genes in the network, increasing the number of genes to 548. The 548 Arabidopsis genes were then used to re-query the Arabidopsis co-expression database and re-construct a network that captured additional network neighbors, expanding to a total of 694 genes. The 694 Arabidopsis genes were computationally divided into 22 clusters. Queries of the Populus genome using the Arabidopsis genes revealed 817 Populus orthologs. Functional analysis of gene ontology and tissue-specific gene expression indicated that these Arabidopsis and Populus genes are high likelihood candidates for functional genomics in relation to cell wall biosynthesis.

  8. SeqEnrich: A tool to predict transcription factor networks from co-expressed Arabidopsis and Brassica napus gene sets.

    Science.gov (United States)

    Becker, Michael G; Walker, Philip L; Pulgar-Vidal, Nadège C; Belmonte, Mark F

    2017-01-01

    Transcription factors and their associated DNA binding sites are key regulatory elements of cellular differentiation, development, and environmental response. New tools that predict transcriptional regulation of biological processes are valuable to researchers studying both model and emerging-model plant systems. SeqEnrich predicts transcription factor networks from co-expressed Arabidopsis or Brassica napus gene sets. The networks produced by SeqEnrich are supported by existing literature and predicted transcription factor-DNA interactions that can be functionally validated at the laboratory bench. The program functions with gene sets of varying sizes and derived from diverse tissues and environmental treatments. SeqEnrich presents as a powerful predictive framework for the analysis of Arabidopsis and Brassica napus co-expression data, and is designed so that researchers at all levels can easily access and interpret predicted transcriptional circuits. The program outperformed its ancestral program ChipEnrich, and produced detailed transcription factor networks from Arabidopsis and Brassica napus gene expression data. The SeqEnrich program is ideal for generating new hypotheses and distilling biological information from large-scale expression data.

  9. [Enhancement of Coprinus cinereus peroxidase in Pichia pastoris by co-expression chaperone PDI and Ero1].

    Science.gov (United States)

    Chen, Fei; Hu, Meirong; Jiang, Xianzhang; Tao, Yong; Huang, Jianzhong

    2015-12-01

    The 1,095 bp gene encoding peroxidase from Coprinus cinereus was synthesized and integrated into the genome of Pichia pastoris with a highly inducible alcohol oxidase. The recombinant CiP (rCiP) fused with the a-mating factor per-pro leader sequence derived from Saccharomyces cerevisiae was secreted into the culture medium and identified as the target protein by mass spectrometry, confirming that a C. cinereus peroxidase (CiP) was successfully expressed in P. pastoris. The endoplasmic reticulum oxidoreductase 1 (Ero1) and protein disulfide isomerase (PDI) were co-expressed with rCiP separately and simultaneously. Compared with the wild type, overexpression of PDI and Erol-PDI increaseed Cip activity in 2.43 and 2.6 fold and their activity reached 316 U/mL and 340 U/mL respectively. The strains co-expressed with Erol-PDI was used to high density fermentation, and their activity reached 3,379 U/mL, which was higher than previously reported of 1,200 U/mL.

  10. Co-expression of estrogen receptor beta and aromatase in Japanese lung cancer patients: gender-dependent clinical outcome.

    Science.gov (United States)

    Verma, Mohit Kumar; Miki, Yasuhiro; Abe, Keiko; Nagasaki, Shuji; Niikawa, Hiromichi; Suzuki, Satoshi; Kondo, Takashi; Sasano, Hironobu

    2012-10-22

    The potential gender differences in lung cancer development have been proposed on the basis of hormonal actions. We aimed to evaluate whether estrogen receptors (ERs) in non-small cell carcinoma (NSCLC) patients may primarily depend upon intratumoral estrogen produced via aromatase pathway. We evaluated ER beta (ERβ) and aromatase status in 169 Japanese NSCLC patients through immunohistochemistry analysis (IHC). Significance of IHC was further confirmed in NSCLC cell lines via in vitro assays. IHC analysis of NSCLC patients demonstrated that both ERβ and aromatase were highly co-expressed (p=0.032) in carcinoma cells. Overall survival in males was significantly worse than that in postmenopausal female among double positive NSCLC patients (p=0.010) but not in non-double positive patients. In addition, among double positive cases, overall survival of males was significantly worse than that of postmenopausal females in those with higher ERβ Allred score ≥5, (p=0.034), but not in those with lower ERβ Allred score=3-4. In-vitro analysis demonstrated aromatase activity on testosterone treatment, which resulted in in situ estrogen production (pblocker i.e. fulvestrant abrogated this effect, (p<0.0001). Our results suggest that co-expression of ERβ and aromatase in NSCLCs of Japanese males may result in tumor progression and potential endocrine therapy may confer therapeutic benefits to these patients. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Matrix factorization reveals aging-specific co-expression gene modules in the fat and muscle tissues in nonhuman primates

    Science.gov (United States)

    Wang, Yongcui; Zhao, Weiling; Zhou, Xiaobo

    2016-10-01

    Accurate identification of coherent transcriptional modules (subnetworks) in adipose and muscle tissues is important for revealing the related mechanisms and co-regulated pathways involved in the development of aging-related diseases. Here, we proposed a systematically computational approach, called ICEGM, to Identify the Co-Expression Gene Modules through a novel mathematical framework of Higher-Order Generalized Singular Value Decomposition (HO-GSVD). ICEGM was applied on the adipose, and heart and skeletal muscle tissues in old and young female African green vervet monkeys. The genes associated with the development of inflammation, cardiovascular and skeletal disorder diseases, and cancer were revealed by the ICEGM. Meanwhile, genes in the ICEGM modules were also enriched in the adipocytes, smooth muscle cells, cardiac myocytes, and immune cells. Comprehensive disease annotation and canonical pathway analysis indicated that immune cells, adipocytes, cardiomyocytes, and smooth muscle cells played a synergistic role in cardiac and physical functions in the aged monkeys by regulation of the biological processes associated with metabolism, inflammation, and atherosclerosis. In conclusion, the ICEGM provides an efficiently systematic framework for decoding the co-expression gene modules in multiple tissues. Analysis of genes in the ICEGM module yielded important insights on the cooperative role of multiple tissues in the development of diseases.

  12. Transcriptomics of the late gestation ovine fetal brain: modeling the co-expression of immune marker genes.

    Science.gov (United States)

    Rabaglino, Maria B; Keller-Wood, Maureen; Wood, Charles E

    2014-11-19

    Major changes in gene expression occur in the fetal brain to modulate the function of this organ postnatally. Thus, factors can alter the genomics of the fetal brain, predisposing to neurological disorders later in life. We hypothesized that the physiological dynamics of the immune system transcriptome of the fetal brain during the last stage of gestation will reveal patterns of immune function and development in the developing brain. In this study we applied weighted gene co-expression analysis of microarrays performed on ovine fetal brain samples, to model the changes in gene expression throughout the second half of gestation. Clusters of co-expressed genes that strongly increase in expression toward the first day of extra-uterine life are related to the hematopoietic lineage, while activation of immune pathways is induced after birth. Moreover, the pattern of gene expression suggests induction of tolerance mechanisms, probably necessary to protect highly produced proteins--such as myelin basic protein--from an autoimmune attack. This study provides insight into the dramatic changes in gene expression that take place in the brain during the fetal life, especially during the last stage of gestation, and suggests that the immune system may have an important role in maturation of the fetal brain, which if disrupted or altered, could have negative consequences in postnatal life.

  13. Integration of Metabolic Modeling with Gene Co-expression Reveals Transcriptionally Programmed Reactions Explaining Robustness in Mycobacterium tuberculosis.

    Science.gov (United States)

    Puniya, Bhanwar Lal; Kulshreshtha, Deepika; Mittal, Inna; Mobeen, Ahmed; Ramachandran, Srinivasan

    2016-03-22

    Robustness of metabolic networks is accomplished by gene regulation, modularity, re-routing of metabolites and plasticity. Here, we probed robustness against perturbations of biochemical reactions of M. tuberculosis in the form of predicting compensatory trends. In order to investigate the transcriptional programming of genes associated with correlated fluxes, we integrated with gene co-expression network. Knock down of the reactions NADH2r and ATPS responsible for producing the hub metabolites, and Central carbon metabolism had the highest proportion of their associated genes under transcriptional co-expression with genes of their flux correlated reactions. Reciprocal gene expression correlations were observed among compensatory routes, fresh activation of alternative routes and in the multi-copy genes of Cysteine synthase and of Phosphate transporter. Knock down of 46 reactions caused the activation of Isocitrate lyase or Malate synthase or both reactions, which are central to the persistent state of M. tuberculosis. A total of 30 new freshly activated routes including Cytochrome c oxidase, Lactate dehydrogenase, and Glycine cleavage system were predicted, which could be responsible for switching into dormant or persistent state. Thus, our integrated approach of exploring transcriptional programming of flux correlated reactions has the potential to unravel features of system architecture conferring robustness.

  14. PDGFRα/HER2 and PDGFRα/p53 Co-expression in Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    Cierpikowski, Piotr; Lis-Nawara, Anna; Gajdzis, Pawel; Bar, Julia

    2018-02-01

    The purpose of this study was to explore the parallel expression of platelet-derived growth factor receptor α (PDGFRα) and human epidermal growth factor receptor 2 (HER2) or p53 in relation to clinicopathological parameters of oral squamous cell carcinoma (OSCC) to define their role in progressive growth of tumor. Expression of PDGFRα, HER2 and p53 was evaluated in 71 OSCC samples by immunohistochemistry. HER2 status was verified by fluorescence in situ hybridization. PDGFRα and p53 expression were associated with tumor grade (p=0.043 and p=0.040, respectively). HER2 expression was more frequent in advanced (III/IV) cancer (p=0.006). A positive correlation of PDGFRα with HER2 (r=0.267; p=0.024) and with p53 (r=0.266; p=0.025) was noted. PDGFRα/HER2 and PDGFRα/p53 co-expression was found more often in G3 than in G1 and G2 tumors (p=0.008 and p=0.015, respectively). Our study revealed that PDGFRα/HER2 and PDGFRα/p53 co-expression exists in poorly differentiated OSCCs, suggesting that cooperation between these proteins might enhance aggressive behavior of tumor. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  15. Intracellular co-expression of Vitreoscilla hemoglobin enhances cell performance and β-galactosidase production in Pichia pastoris.

    Science.gov (United States)

    Wu, Jyh-Ming; Fu, Wei-Chang

    2012-03-01

    Pichia pastoris has been used to produce various recombinant proteins under high oxygen demand conditions. To improve the heterologous production of β-galactosidase, the vgb gene encoding Vitreoscilla hemoglobin (VHb) was co-expressed in the P. pastoris cytoplasm under the control of the methanol-inducible promoter. Co-expression of VHb under different aeration conditions improved cell performance in terms of growth, viability, respiratory rate, and β-galactosidase production. Under limiting aeration conditions, the VHb(+) strain produced 28.2% more biomass but 31.2% less total β-galactosidase activity than the VHb(-) strain. Under non-limiting aeration conditions, the VHb(+) strain showed 20.3% higher cell growth and 9.9% more total β-galactosidase activity than the VHb(-) strain. Moreover, under these conditions, the VHb(+) strain was 7.7% more viable and had a 28.2% higher oxygen uptake rate (OUR) than the VHb(-) strain. Evidently, VHb can enhance the OUR and promote methanol metabolism, thereby improving cell performance and β-galactosidase production. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. Differential co-expression and regulation analyses reveal different mechanisms underlying major depressive disorder and subsyndromal symptomatic depression.

    Science.gov (United States)

    Xu, Fan; Yang, Jing; Chen, Jin; Wu, Qingyuan; Gong, Wei; Zhang, Jianguo; Shao, Weihua; Mu, Jun; Yang, Deyu; Yang, Yongtao; Li, Zhiwei; Xie, Peng

    2015-04-03

    Recent depression research has revealed a growing awareness of how to best classify depression into depressive subtypes. Appropriately subtyping depression can lead to identification of subtypes that are more responsive to current pharmacological treatment and aid in separating out depressed patients in which current antidepressants are not particularly effective. Differential co-expression analysis (DCEA) and differential regulation analysis (DRA) were applied to compare the transcriptomic profiles of peripheral blood lymphocytes from patients with two depressive subtypes: major depressive disorder (MDD) and subsyndromal symptomatic depression (SSD). Six differentially regulated genes (DRGs) (FOSL1, SRF, JUN, TFAP4, SOX9, and HLF) and 16 transcription factor-to-target differentially co-expressed gene links or pairs (TF2target DCLs) appear to be the key differential factors in MDD; in contrast, one DRG (PATZ1) and eight TF2target DCLs appear to be the key differential factors in SSD. There was no overlap between the MDD target genes and SSD target genes. Venlafaxine (Efexor™, Effexor™) appears to have a significant effect on the gene expression profile of MDD patients but no significant effect on the gene expression profile of SSD patients. DCEA and DRA revealed no apparent similarities between the differential regulatory processes underlying MDD and SSD. This bioinformatic analysis may provide novel insights that can support future antidepressant R&D efforts.

  17. Co-expression of tyrosine hydroxylase and glutamic acid decarboxylase in dopamine differentiation factor-treated striatal neurons in culture.

    Science.gov (United States)

    Max, S R; Bossio, A; Iacovitti, L

    1996-01-22

    We have previously shown that dopamine differentiation factors (DDF) can stimulate the novel expression of tyrosine hydroxylase (TH) in the phenotypically plastic neurons of the embryonic mouse striatum (Du et al., J. Neurosci., 14 (1994) 7688-7694; Du and Iacovitti, J. Neurosci., 15 (1995) 5420-5427). The present study sought to determine whether TH induction required down-regulation of an existing GABAergic trait in striatal neurons or whether enzymes of both neurotransmitter systems were simultaneously expressed. Immunocytochemical analysis revealed that, following treatment with DDFs, TH and the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) were co-expressed in the same neurons. Moreover, GAD enzyme activity was not affected by the dramatic increase in TH. Thus, the induction of a novel neurotransmitter phenotype in brain neurons does not appear to occur at the expense of the existing phenotype.

  18. Transcription factor-pathway co-expression analysis reveals cooperation between SP1 and ESR1 on dysregulating cell cycle arrest in non-hyperdiploid multiple myeloma

    Science.gov (United States)

    Wang, Xujun; Yan, Zhenyu; Fulciniti, Mariateresa; Li, Yingxiang; Gkotzamanidou, Maria; Amin, Samir B; Shah, Parantu K; Zhang, Yong

    2014-01-01

    Multiple myeloma is a hematological cancer of plasma B-cells and remains incurable. Two major subtypes of myeloma, hyperdiploid (HMM) and non-hyperdiploid myeloma (NHMM), have distinct chromosomal alterations and different survival outcomes. Transcription factors (TrFs) have been implicated in myeloma oncogenesis but their dysregulation in myeloma subtypes are less studied. Here we develop a TrF-pathway co-expression analysis to identify altered co-expression between two sample types. We apply the method to the two myeloma subtypes and the cell cycle arrest pathway, which is significantly differentially expressed between the two subtypes. We find that TrFs MYC, NF-κB and HOXA9 have significantly lower co-expression with cell cycle arrest in HMM, co-occurring with their over-activation in HMM. In contrast, TrFs ESR1, SP1 and E2F1 have significantly lower co-expression with cell cycle arrest in NHMM. SP1 ChIP targets are enriched by cell cycle arrest genes. These results motivate a cooperation model of ESR1 and SP1 in regulating cell cycle arrest, and a hypothesis that their over-activation in NHMM disrupts proper regulation of cell cycle arrest. Co-targeting ESR1 and SP1 shows a synergistic effect on inhibiting myeloma proliferation in NHMM cell lines. Therefore, studying TrF-pathway co-expression dysregulation in human cancers facilitates forming novel hypotheses towards clinical utility. PMID:23925045

  19. Co-Expression of TWIST1 and ZEB2 in Oral Squamous Cell Carcinoma Is Associated with Poor Survival.

    Directory of Open Access Journals (Sweden)

    Yink Heay Kong

    Full Text Available Oral squamous cell carcinoma (OSCC is an aggressive disease accounting for more than 260,000 cancer cases diagnosed and 128,000 deaths worldwide. A large majority of cancer deaths result from cancers that have metastasized beyond the primary tumor. The relationship between genetic changes and clinical outcome can reflect the biological events that promote cancer's aggressive behavior, and these can serve as molecular markers for improved patient management and survival. To this end, epithelial-mesenchymal transition (EMT is a major process that promotes tumor invasion and metastasis, making EMT-related proteins attractive diagnostic biomarkers and therapeutic targets. In this study, we used immunohistochemistry to study the expression of a panel of transcription factors (TWIST1, SNAI1/2, ZEB1 and ZEB2 and other genes intimately related to EMT (CDH1 and LAMC2 at the invasive tumor front of OSCC tissues. The association between the expression of these proteins and clinico-pathological parameters were examined with Pearson Chi-square and correlation with survival was analyzed using Kaplan Meier analysis. Our results demonstrate that there was a significant differential expression of CDH1, LAMC2, SNAI1/2 and TWIST1 between OSCC and normal oral mucosa (NOM. Specifically, CDH1 loss was significantly associated with Broder's grading, while diffused LAMC2 was similarly associated with non-cohesive pattern of invasion. Notably, co-expression of TWIST1 and ZEB2 in OSCC was significantly associated with poorer overall survival, particularly in patients without detectable lymph node metastasis. This study demonstrates that EMT-related proteins are differentially expressed in OSCC and that the co-expression of TWIST1 and ZEB2 could be of clinical value in identifying patients with poor survival for appropriate patient management.

  20. Transcriptome-wide co-expression analysis identifies LRRC2 as a novel mediator of mitochondrial and cardiac function.

    Directory of Open Access Journals (Sweden)

    Chris McDermott-Roe

    Full Text Available Mitochondrial dysfunction contributes to myriad monogenic and complex pathologies. To understand the underlying mechanisms, it is essential to define the full complement of proteins that modulate mitochondrial function. To identify such proteins, we performed a meta-analysis of publicly available gene expression data. Gene co-expression analysis of a large and heterogeneous compendium of microarray data nominated a sub-population of transcripts that whilst highly correlated with known mitochondrial protein-encoding transcripts (MPETs, are not themselves recognized as generating proteins either localized to the mitochondrion or pertinent to functions therein. To focus the analysis on a medically-important condition with a strong yet incompletely understood mitochondrial component, candidates were cross-referenced with an MPET-enriched module independently generated via genome-wide co-expression network analysis of a human heart failure gene expression dataset. The strongest uncharacterized candidate in the analysis was Leucine Rich Repeat Containing 2 (LRRC2. LRRC2 was found to be localized to the mitochondria in human cells and transcriptionally-regulated by the mitochondrial master regulator Pgc-1α. We report that Lrrc2 transcript abundance correlates with that of β-MHC, a canonical marker of cardiac hypertrophy in humans and experimentally demonstrated an elevation in Lrrc2 transcript in in vitro and in vivo rodent models of cardiac hypertrophy as well as in patients with dilated cardiomyopathy. RNAi-mediated Lrrc2 knockdown in a rat-derived cardiomyocyte cell line resulted in enhanced expression of canonical hypertrophic biomarkers as well as increased mitochondrial mass in the context of increased Pgc-1α expression. In conclusion, our meta-analysis represents a simple yet powerful springboard for the nomination of putative mitochondrially-pertinent proteins relevant to cardiac function and enabled the identification of LRRC2 as a novel

  1. Identifying the optimal gene and gene set in hepatocellular carcinoma based on differential expression and differential co-expression algorithm.

    Science.gov (United States)

    Dong, Li-Yang; Zhou, Wei-Zhong; Ni, Jun-Wei; Xiang, Wei; Hu, Wen-Hao; Yu, Chang; Li, Hai-Yan

    2017-02-01

    The objective of this study was to identify the optimal gene and gene set for hepatocellular carcinoma (HCC) utilizing differential expression and differential co-expression (DEDC) algorithm. The DEDC algorithm consisted of four parts: calculating differential expression (DE) by absolute t-value in t-statistics; computing differential co-expression (DC) based on Z-test; determining optimal thresholds on the basis of Chi-squared (χ2) maximization and the corresponding gene was the optimal gene; and evaluating functional relevance of genes categorized into different partitions to determine the optimal gene set with highest mean minimum functional information (FI) gain (Δ*G). The optimal thresholds divided genes into four partitions, high DE and high DC (HDE-HDC), high DE and low DC (HDE-LDC), low DE and high DC (LDE‑HDC), and low DE and low DC (LDE-LDC). In addition, the optimal gene was validated by conducting reverse transcription-polymerase chain reaction (RT-PCR) assay. The optimal threshold for DC and DE were 1.032 and 1.911, respectively. Using the optimal gene, the genes were divided into four partitions including: HDE-HDC (2,053 genes), HED-LDC (2,822 genes), LDE-HDC (2,622 genes), and LDE-LDC (6,169 genes). The optimal gene was microtubule‑associated protein RP/EB family member 1 (MAPRE1), and RT-PCR assay validated the significant difference between the HCC and normal state. The optimal gene set was nucleoside metabolic process (GO\\GO:0009116) with Δ*G = 18.681 and 24 HDE-HDC partitions in total. In conclusion, we successfully investigated the optimal gene, MAPRE1, and gene set, nucleoside metabolic process, which may be potential biomarkers for targeted therapy and provide significant insight for revealing the pathological mechanism underlying HCC.

  2. Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

    Directory of Open Access Journals (Sweden)

    Hudler Petra

    2009-10-01

    Full Text Available Abstract Background Loss of DNA mismatch repair (MMR in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC. Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells. Methods Yeast MLH1 and PMS1 genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested. Results The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic. Conclusion Results of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene.

  3. Co-expression of sulphydryl oxidase and protein disulphide isomerase in Escherichia coli allows for production of soluble CRM197.

    Science.gov (United States)

    Roth, R; van Zyl, P; Tsekoa, T; Stoychev, S; Mamputha, S; Buthelezi, S; Crampton, M

    2017-05-01

    To investigate the production of soluble cross-reacting material 197 (CRM197 ) in Escherichia coli, a safe and effective T-cell-dependent protein carrier for polysaccharides used in the manufacture and application of multivalent conjugate vaccines. The use of co-expression of a sulphydryl oxidase (SOX) and protein disulphide isomerase for the production of soluble CRM197 in E. coli is described. CRM197 contains two disulphide bonds, which are normally unable to form in the reducing environment of the E. coli cytoplasm. It was found that co-expression yielded soluble CRM197 , at a production rate ~10% of the production of insoluble CRM197 , in equivalent small-scale cultures. Structural analysis of the purified CRM197 compared to CRM197 commercially produced in cultures of recombinant Pseudomonas fluorescens indicated that the E. coli soluble protein compares favourably on all structural levels. SOX and protein disulphide isomerase are enzymes involved in the formation of intra-protein disulphide bonds, and can influence the tertiary structure of the protein being produced, resulting in increased solubility due to the correct folding of the protein. Their use enabled the production of soluble untagged CRM197 in E. coli, which was previously unachievable. Previous literature reports have shown that CRM197 can be expressed in E. coli, though only in an insoluble form, or in soluble form as a fusion protein. It is currently commercially produced in cultures of recombinant P. fluorescens. The use of a widely used, well-characterized expression host such as E. coli, rather than P. fluorescens broadens the applicability of the production technology, and the production system described here is worthy of further investigation for scaled up manufacture of CRM197 . © 2017 The Society for Applied Microbiology.

  4. ConGEMs: Condensed Gene Co-Expression Module Discovery Through Rule-Based Clustering and Its Application to Carcinogenesis

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    Saurav Mallik

    2017-12-01

    Full Text Available For transcriptomic analysis, there are numerous microarray-based genomic data, especially those generated for cancer research. The typical analysis measures the difference between a cancer sample-group and a matched control group for each transcript or gene. Association rule mining is used to discover interesting item sets through rule-based methodology. Thus, it has advantages to find causal effect relationships between the transcripts. In this work, we introduce two new rule-based similarity measures—weighted rank-based Jaccard and Cosine measures—and then propose a novel computational framework to detect condensed gene co-expression modules ( C o n G E M s through the association rule-based learning system and the weighted similarity scores. In practice, the list of evolved condensed markers that consists of both singular and complex markers in nature depends on the corresponding condensed gene sets in either antecedent or consequent of the rules of the resultant modules. In our evaluation, these markers could be supported by literature evidence, KEGG (Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology annotations. Specifically, we preliminarily identified differentially expressed genes using an empirical Bayes test. A recently developed algorithm—RANWAR—was then utilized to determine the association rules from these genes. Based on that, we computed the integrated similarity scores of these rule-based similarity measures between each rule-pair, and the resultant scores were used for clustering to identify the co-expressed rule-modules. We applied our method to a gene expression dataset for lung squamous cell carcinoma and a genome methylation dataset for uterine cervical carcinogenesis. Our proposed module discovery method produced better results than the traditional gene-module discovery measures. In summary, our proposed rule-based method is useful for exploring biomarker modules from transcriptomic data.

  5. Improved 1, 2, 4-butanetriol production from an engineered Escherichia coli by co-expression of different chaperone proteins.

    Science.gov (United States)

    Lu, Xinyao; He, Shuying; Zong, Hong; Song, Jian; Chen, Wen; Zhuge, Bin

    2016-09-01

    1, 2, 4-Butanetriol (BT) is a high-value non-natural chemical and has important applications in polymers, medical production and military industry. In the constructed BT biosynthesis pathway from xylose in Escherichia coli, the xylose dehydrogenase (Xdh) and the benzoylformate decarboxylase (MdlC) are heterologous enzymes and the activity of MdlC is the key limiting factor for BT production. In this study, six chaperone protein systems were introduced into the engineered E. coli harboring the recombinant BT pathway. The chaperone GroES-GroEL was beneficial to Xdh activity but had a negative effect on MdlC activity and BT titer. The plasmid pTf16 containing the tig gene (trigger factor) was beneficial to Xdh and MdlC activities and improved the BT titer from 0.42 to 0.56 g/l from 20 g/l xylose. However, co-expression of trigger factor and GroES-GroEL simultaneously reduced the activity of MdlC and had no effect on the BT production. The plasmid pKJE7 harboring dnaK-dnaJ-grpE showed significant negative effects on these enzyme activities and cell growth, leading to completely restrained the BT production. Similarly, co-expression of DnaKJ-GrpPE and GroES-GroEL simultaneously reduced Xdh and MdlC activities and decreased the BT titer by 45.2 %. The BT production of the engineered E. coli harboring pTf16 was further improved to the highest level at 1.01 g/l under pH control (pH 7). This work showed the potential application of chaperone proteins in microorganism engineering to get high production of target compounds as an effective and valuable tool.

  6. Neurotensin Is Co-Expressed, Co-Released And Acts Together With Glp-1 And Pyy In Enteroendocrine Control Of Metabolism

    DEFF Research Database (Denmark)

    Grunddal, Kaare Villum; Ratner, Cecilia F; Svendsen, Berit

    2016-01-01

    The two gut hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are well known to be co-expressed, co-stored and released together to co-act in the control of key metabolic target organs. However, recently it became clear that several other gut hormones can be co-expressed in the intest......The two gut hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are well known to be co-expressed, co-stored and released together to co-act in the control of key metabolic target organs. However, recently it became clear that several other gut hormones can be co...... deeply integrated with GLP-1 and PYY, which should be taken into account when exploiting the enteroendocrine regulation of metabolism pharmacologically....

  7. Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3/phospho-Tau interaction.

    Science.gov (United States)

    Tugaeva, Kristina V; Tsvetkov, Philipp O; Sluchanko, Nikolai N

    2017-01-01

    Abundant regulatory 14-3-3 proteins have an extremely wide interactome and coordinate multiple cellular events via interaction with specifically phosphorylated partner proteins. Notwithstanding the key role of 14-3-3/phosphotarget interactions in many physiological and pathological processes, they are dramatically underexplored. Here, we focused on the 14-3-3 interaction with human Tau protein associated with the development of several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Among many known phosphorylation sites within Tau, protein kinase A (PKA) phosphorylates several key residues of Tau and induces its tight interaction with 14-3-3 proteins. However, the stoichiometry and mechanism of 14-3-3 interaction with phosphorylated Tau (pTau) are not clearly elucidated. In this work, we describe a simple bacterial co-expression system aimed to facilitate biochemical and structural studies on the 14-3-3/pTau interaction. We show that dual co-expression of human fetal Tau with PKA in Escherichia coli results in multisite Tau phosphorylation including also naturally occurring sites which were not previously considered in the context of 14-3-3 binding. Tau protein co-expressed with PKA displays tight functional interaction with 14-3-3 isoforms of a different type. Upon triple co-expression with 14-3-3 and PKA, Tau protein could be co-purified with 14-3-3 and demonstrates complex which is similar to that formed in vitro between individual 14-3-3 and pTau obtained from dual co-expression. Although used in this study for the specific case of the previously known 14-3-3/pTau interaction, our co-expression system may be useful to study of other selected 14-3-3/phosphotarget interactions and for validations of 14-3-3 complexes identified by other methods.

  8. Mycobacterium tuberculosis chaperonin 10 and N-truncated fragments. Their synthesis and purification by the isoelectric focusing technique carried out in solution.

    Science.gov (United States)

    Lucietto, P; Fossati, G; Ball, H L; Giuliani, P; Mascagni, P

    1997-04-01

    The Mycobacterium tuberculosis chaperonin 10 protein and fragments corresponding to sequences 59-99, 51-99 and 26-99 were synthesised by the solid-phase methodology using a double coupling protocol and without the aid of capping agents. After the final acid cleavage using the low TFMSA-high HF protocol the polypeptides were purified by either the ion exchange chromatography/RP-HPLC combination or the isoelectric separation carried out in solution and followed by semi-preparative RP-HPLC. Comparison of the results obtained through the two approaches indicated that in general the isoelectricfocusing/HPLC combination was superior both in terms of recovery of final material and its purity. The advantages found were as follows: (i) Unlike ion exchange chromatography, no tailoring of the separation conditions is required, (ii) Several consecutive focusings can be carried out in progressively narrower pH gradients. This increases the separation resolution without the need of changing other separation parameters, (iii) Very little manipulation is needed, and each focusing requires 3-5h. (iv) Full compatibility with non-ionic denaturants such as 8 M urea. This increases solubility so that using the ROTOFOR instrument described here 50-100 mg crude polypeptide can be processed daily. Thus the isoelectric focusing technique carried out in solution is a valid and inexpensive alternative to ion exchange chromatography.

  9. Chaperonin containing T-complex polypeptide subunit eta is a potential marker of joint contracture: an experimental study in the rat.

    Science.gov (United States)

    He, Ronghan; Wang, Zhe; Lu, Yunxiang; Huang, Junqi; Ren, Jianhua; Wang, Kun

    2015-11-01

    Joint contracture is a fibroproliferative disorder that restricts joint mobility, resulting in tissue degeneration and deformity. However, the etiology of joint contracture is still unknown. Chaperonin containing T-complex polypeptide subunit eta (CCT-eta) is reported to increase in fibrotic diseases. The purpose of this study was to investigate whether CCT-eta is implicated in joint contracture and to determine the role of CCT-eta in the progression of joint contracture by analyzing a rat model. We immobilized the left knee joint of rat by internal fixation for 8 weeks. The non-immobilized right leg served as a control. The range of motion (ROM) of the knee was investigated. Fibroblasts were obtained from the posterior joint capsule of the joints. The outcome was followed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, fibroblast migration assay, and collagen assay. The effect of CCT-eta on the functions of fibroblasts was observed by utilizing a short inhibitory RNA (siRNA) targeting CCT-eta. The ROM of the immobilized joints was significantly limited compared to the contralateral joints (p contracture disease.

  10. Chaperonin-Containing t-Complex Protein-1 Subunit β as a Possible Biomarker for the Phase of Glomerular Hyperfiltration of Diabetic Nephropathy

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    Chung-Ze Wu

    2015-01-01

    Full Text Available In cell model, we discovered the association between chaperonin-containing t-complex polypeptide 1 subunit β (TCP-1β and early diabetic nephropathy (DN. In this study, we further explored the relationships between TCP-1β and type 2 diabetic mellitus (DM. To mimic the clinical hyperfiltration state, a type 2 DM mice model was established by feeding a high-fat diet in combination with treatment of streptozotocin and nicotinamide. Blood and urine were collected to determine creatinine clearance (Ccr, and kidney tissues were harvested for evaluation of TCP-1β expression by immunohistochemistry and Western blot. Meanwhile, clinical subjects of healthy controls and type 2 DM were recruited to strengthen the evidence with urine TCP-1β. Results showed that Ccr and the expression of TCP-1β in kidney were significantly higher one week after hyperglycemia development, suggesting that the hyperfiltration state was successfully established in the mice model. TCP-1β was expressed predominantly on renal tubules. By using the estimated glomerular filtration rate to index progression in clinical investigation, urine TCP-1β level was associated with the hyperfiltration phase in type 2 DM patients. Conclusively, we confirmed that TCP-1β is a possible biomarker for early nephropathy of type 2 DM, but further mechanistic study to elucidate its cause and pathway is needed.

  11. Co-expression of Argonaute2 enhances short hairpin RNA-induced RNA interference in Xenopus CNS neurons in vivo

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    Chih-ming Chen

    2009-07-01

    Full Text Available RNA interference (RNAi is an evolutionarily conserved mechanism for sequence-specific gene silencing. Recent advances in our understanding of RNAi machinery make it possible to reduce protein expression by introducing short hairpin RNA (shRNA into cells of many systems, however, the efficacy of RNAi-mediated protein knockdown can be quite variable, especially in intact animals, and this limits its application. We built adaptable molecular tools, pSilencer (pSi and pReporter (pRe constructs, to evaluate the impact of different promoters, shRNA structures and overexpression of Ago2, the key enzyme in the RNA-induced silencing complex (RISC, on the efficiency of RNAi. The magnitude of RNAi knockdown was evaluated in cultured cells and intact animals by comparing fluorescence intensity levels of GFP, the RNAi target, relative to mCherry, which was not targeted. Co-expression of human Ago2 with shRNA significantly enhanced efficiency of GFP knockdown in cell lines and in neurons of intact Xenopus tadpoles. Human H1- and U6-promotors alone or the U6-promotor with an enhancer element were equally effective at driving GFP knockdown. shRNA derived from the microRNA-30 design (shRNAmir30 enhanced the efficiency of GFP knockdown. Expressing pSi containing Ago2 with shRNA increased knockdown efficiency of an endogenous neuronal protein, the GluR2 subunit of the AMPA receptor, functionally accessed by recording AMPA receptor-mediated spontaneous synaptic currents in Xenopus CNS neurons. Our data suggest that co-expression of Ago2 and shRNA is a simple method to enhance RNAi in intact animals. While morpholino antisense knockdown is effective in Xenopus and Zebrafish, a principle advantage of the RNAi method is the possibility of spatial and temporal control of protein knockdown by use of cell type specific and regulatable pol II promoters to drive shRNA and Ago2. This should extend the application of RNAi to study gene function of intact brain circuits.

  12. Long-term safety and stability of angiogenesis induced by balanced single-vector co-expression of PDGF-BB and VEGF164 in skeletal muscle

    Science.gov (United States)

    Gianni-Barrera, Roberto; Burger, Maximilian; Wolff, Thomas; Heberer, Michael; Schaefer, Dirk J.; Gürke, Lorenz; Mujagic, Edin; Banfi, Andrea

    2016-01-01

    Therapeutic angiogenesis by growth factor delivery is an attractive treatment strategy for ischemic diseases, yet clinical efficacy has been elusive. The angiogenic master regulator VEGF-A can induce aberrant angiogenesis if expressed above a threshold level. Since VEGF remains localized in the matrix around expressing cells, homogeneous dose distribution in target tissues is required, which is challenging. We found that co-expression of the pericyte-recruiting factor PDGF-BB at a fixed ratio with VEGF from a single bicistronic vector ensured normal angiogenesis despite heterogeneous high VEGF levels. Taking advantage of a highly controlled gene delivery platform, based on monoclonal populations of transduced myoblasts, in which every cell stably produces the same amount of each factor, here we rigorously investigated a) the dose-dependent effects, and b) the long-term safety and stability of VEGF and PDGF-BB co-expression in skeletal muscle. PDGF-BB co-expression did not affect the normal angiogenesis by low and medium VEGF doses, but specifically prevented vascular tumors by high VEGF, yielding instead normal and mature capillary networks, accompanied by robust arteriole formation. Induced angiogenesis persisted unchanged up to 4 months, while no tumors appeared. Therefore, PDGF-BB co-expression is an attractive strategy to improve safety and efficacy of therapeutic angiogenesis by VEGF gene delivery. PMID:26882992

  13. Balancing protein similarity and gene co-expression reveals new links between genetic conservation and developmental diversity in invertebrates.

    Science.gov (United States)

    Lefebvre, Céline; Aude, Jean-Christophe; Glémet, Eric; Néri, Christian

    2005-04-15

    To identify genetic conservation relative to precise aspects of developmental diversity, an essential question in computational biology, we developed a new comparative method that allows conserved modules for the best balance between protein sequence similarity and gene co-expression to be constructed, in invertebrates. Our method, referred to as the best-balance constraint procedure (BBCP), yielded 719 functionally conserved modules (FCMs) comprising 2-23 gene pairs. These modules were consistent with the developmental roles of orthologues as inferred from Gene Ontology, RNAi knockouts, InterPro and process-specific microarray data. New relationships were defined between genetic conservation and developmental diversity. Novel gene associations were indeed found in 94% of the FCMs, 150 modules being completely new. A significant proportion of the FCMs (18%, 132 modules) described cell type-specific mechanisms, comprising neuronal, muscle and germ cell signaling, new associations being found in 125 modules. Also found were gene associations for cell fate specification activities previously not highlighted by computational means, e.g. in FCMs containing homeogenes. These data indicate that highly discriminative description of genetic conservation can be deduced using BBCP, and reveal new correlations between cellular and developmental diversity and gene essentiality in invertebrates. christian.neri@broca.inserm.fr For supplementary information, please refer to Bioinformatics online.

  14. Co-expression of sialic acid receptors compatible with avian and human influenza virus binding in emus (Dromaius novaehollandiae).

    Science.gov (United States)

    Gujjar, Naveen; Chothe, Shubhada K; Gawai, Shashikant; Nissly, Ruth; Bhushan, Gitanjali; Kanagaraj, Vijayarani; Jayarao, Bhushan M; Kathaperumal, Kumanan; Subbiah, Madhuri; Kuchipudi, Suresh V

    2017-01-01

    Influenza A viruses (IAVs) continue to threaten animal and human health with constant emergence of novel variants. While aquatic birds are a major reservoir of most IAVs, the role of other terrestrial birds in the evolution of IAVs is becoming increasingly evident. Since 2006, several reports of IAV isolations from emus have surfaced and avian influenza infection of emus can lead to the selection of mammalian like PB2-E627K and PB2-D701N mutants. However, the potential of emus to be co-infected with avian and mammalian IAVs is not yet understood. As a first step, we investigated sialic acid (SA) receptor distribution across major organs and body systems of emu and found a widespread co-expression of both SAα2,3Gal and SAα2,6Gal receptors in various tissues that are compatible with avian and human IAV binding. Our results suggest that emus could allow genetic recombination and hence play an important role in the evolution of IAVs. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Resilient protein co-expression network in male orbitofrontal cortex layer 2/3 during human aging.

    Science.gov (United States)

    Pabba, Mohan; Scifo, Enzo; Kapadia, Fenika; Nikolova, Yuliya S; Ma, Tianzhou; Mechawar, Naguib; Tseng, George C; Sibille, Etienne

    2017-10-01

    The orbitofrontal cortex (OFC) is vulnerable to normal and pathologic aging. Currently, layer resolution large-scale proteomic studies describing "normal" age-related alterations at OFC are not available. Here, we performed a large-scale exploratory high-throughput mass spectrometry-based protein analysis on OFC layer 2/3 from 15 "young" (15-43 years) and 18 "old" (62-88 years) human male subjects. We detected 4193 proteins and identified 127 differentially expressed (DE) proteins (p-value ≤0.05; effect size >20%), including 65 up- and 62 downregulated proteins (e.g., GFAP, CALB1). Using a previously described categorization of biological aging based on somatic tissues, that is, peripheral "hallmarks of aging," and considering overlap in protein function, we show the highest representation of altered cell-cell communication (54%), deregulated nutrient sensing (39%), and loss of proteostasis (35%) in the set of OFC layer 2/3 DE proteins. DE proteins also showed a significant association with several neurologic disorders; for example, Alzheimer's disease and schizophrenia. Notably, despite age-related changes in individual protein levels, protein co-expression modules were remarkably conserved across age groups, suggesting robust functional homeostasis. Collectively, these results provide biological insight into aging and associated homeostatic mechanisms that maintain normal brain function with advancing age. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Gene Co-Expression Network Analysis Unraveling Transcriptional Regulation of High-Altitude Adaptation of Tibetan Pig.

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    Cunling Jia

    Full Text Available Tibetan pigs have survived at high altitude for millennia and they have a suite of adaptive features to tolerate the hypoxic environment. However, the molecular mechanisms underlying the regulation of hypoxia-adaptive phenotypes have not been completely elucidated. In this study, we analyzed differentially expressed genes (DEGs, biological pathways and constructed co-expression regulation networks using whole-transcriptome microarrays from lung tissues of Tibetan and Duroc pigs both at high and low altitude. A total of 3,066 DEGs were identified and this list was over-represented for the ontology terms including metabolic process, catalytic activity, and KEGG pathway including metabolic pathway and PI3K-Akt signaling pathway. The regulatory (RIF and phenotypic (PIF impact factor analysis identified several known and several potentially novel regulators of hypoxia adaption, including: IKBKG, KLF6 and RBPJ (RIF1, SF3B1, EFEMP1, HOXB6 and ATF6 (RIF2. These findings provide new details of the regulatory architecture of hypoxia-adaptive genes and also insight into which genes may undergo epigenetic modification for further study in the high-altitude adaptation.

  17. Human breast cancer cell lines co-express neuronal, epithelial, and melanocytic differentiation markers in vitro and in vivo.

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    Qingbei Zhang

    Full Text Available Differentiation programs are aberrant in cancer cells allowing them to express differentiation markers in addition to their tissue of origin. In the present study, we demonstrate the multi-lineage differentiation potential of breast cancer cell lines to express multiple neuronal/glial lineage-specific markers as well as mammary epithelial and melanocytic-specific markers. Multilineage expression was detected in luminal (MCF-7 and SKBR3 and basal (MDA-MB-231 types of human breast cancer cell lines. We also observed comparable co-expression of these three cell lineage markers in MDA-MB-435 cells in vitro, in MDA-MB-435 primary tumors derived from parental and single cell clones and in lung metastases in vivo. Furthermore, ectoderm multi-lineage transdifferentiation was also found in human melanoma (Ul-MeL and glioblastoma cell lines (U87 and D54. These observations indicate that aberrant multi-lineage transdifferentiation or lineage infidelity may be a wide spread phenomenon in cancer.

  18. Co-expression of two subtypes of melatonin receptor on rat M1-type intrinsically photosensitive retinal ganglion cells.

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    Wen-Long Sheng

    Full Text Available Intrinsically photosensitive retinal ganglion cells (ipRGCs are involved in circadian and other non-image forming visual responses. An open question is whether the activity of these neurons may also be under the regulation mediated by the neurohormone melatonin. In the present work, by double-staining immunohistochemical technique, we studied the expression of MT1 and MT2, two known subtypes of mammalian melatonin receptors, in rat ipRGCs. A single subset of retinal ganglion cells labeled by the specific antibody against melanopsin exhibited the morphology typical of M1-type ipRGCs. Immunoreactivity for both MT1 and MT2 receptors was clearly seen in the cytoplasm of all labeled ipRGCs, indicating that these two receptors were co-expressed in each of these neurons. Furthermore, labeling for both the receptors were found in neonatal M1 cells as early as the day of birth. It is therefore highly plausible that retinal melatonin may directly modulate the activity of ipRGCs, thus regulating non-image forming visual functions.

  19. In planta production of the highly potent resveratrol analogue pterostilbene via stilbene synthase and O-methyltransferase co-expression

    Energy Technology Data Exchange (ETDEWEB)

    Rimando A. M.; Liu C.; Pan, Z.; Polashock, J. J.; Dayan, F. E., Mizuno, C. S.; Snook, M. E.; Baerson, S. R.

    2012-04-01

    Resveratrol and related stilbenes are thought to play important roles in defence responses in several plant species and have also generated considerable interest as nutraceuticals owing to their diverse health-promoting properties. Pterostilbene, a 3,5-dimethylether derivative of resveratrol, possesses properties similar to its parent compound and, additionally, exhibits significantly higher fungicidal activity in vitro and superior pharmacokinetic properties in vivo. Recombinant enzyme studies carried out using a previously characterized O-methyltransferase sequence from Sorghum bicolor (SbOMT3) demonstrated its ability to catalyse the A ring-specific 3,5-bis-O-methylation of resveratrol, yielding pterostilbene. A binary vector was constructed for the constitutive co-expression of SbOMT3 with a stilbene synthase sequence from peanut (AhSTS3) and used for the generation of stably transformed tobacco and Arabidopsis plants, resulting in the accumulation of pterostilbene in both species. A reduced floral pigmentation phenotype observed in multiple tobacco transformants was further investigated by reversed-phase HPLC analysis, revealing substantial decreases in both dihydroquercetin-derived flavonoids and phenylpropanoid-conjugated polyamines in pterostilbene-producing SbOMT3/AhSTS3 events. These results demonstrate the potential utility of this strategy for the generation of pterostilbene-producing crops and also underscore the need for the development of additional approaches for minimizing concomitant reductions in key phenylpropanoid-derived metabolites.

  20. Cochlin induced TREK-1 co-expression and annexin A2 secretion: role in trabecular meshwork cell elongation and motility.

    Science.gov (United States)

    Goel, Manik; Sienkiewicz, Adam E; Picciani, Renata; Lee, Richard K; Bhattacharya, Sanjoy K

    2011-01-01

    Fluid flow through large interstitial spaces is sensed at the cellular level, and mechanistic responses to flow changes enables expansion or contraction of the cells modulating the surrounding area and brings about changes in fluid flow. In the anterior eye chamber, aqueous humor, a clear fluid, flows through trabecular meshwork (TM), a filter like region. Cochlin, a secreted protein in the extracellular matrix, was identified in the TM of glaucomatous patients but not controls by mass spectrometry. Cochlin undergoes shear induced multimerization and plays a role in mechanosensing of fluid shear. Cytoskeletal changes in response to mechanosensing in the ECM by cochlin will necessitate transduction of mechanosensing. TREK-1, a stretch activated outward rectifying potassium channel protein known to act as mechanotransducer was found to be expressed in TM. Cochlin expression results in co-expression of TREK-1 and filopodia formation. Prolonged cochlin expression results in expression and subsequent secretion of annexin A2, a protein known to play a role in cytoskeletal remodeling. Cochlin interacts with TREK-1 and annexin A2. Cochlin-TREK-1 interaction has functional consequences and results in changes in cell shape and motility. Annexin A2 expression and secretion follows cochlin-TREK-1 syn-expression and correlates with cell elongation. Thus cytoskeleton changes in response to fluid shear sensed by cochlin are further mediated by TREK-1 and annexin A2.

  1. Development of stable Vibrio cholerae O1 Hikojima type vaccine strains co-expressing the Inaba and Ogawa lipopolysaccharide antigens.

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    Stefan L Karlsson

    Full Text Available We describe here the development of stable classical and El Tor V. cholerae O1 strains of the Hikojima serotype that co-express the Inaba and Ogawa antigens of O1 lipopolysaccharide (LPS. Mutation of the wbeT gene reduced LPS perosamine methylation and thereby gave only partial transformation into Ogawa LPS on the cell surface. The strains express approximately equal amounts of Inaba- and Ogawa-LPS antigens which are preserved after formalin-inactivation of the bacteria. Oral immunizations of both inbred and outbred mice with formalin-inactivated whole-cell vaccine preparations of these strains elicited strong intestinal IgA anti-LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine. Passive protection studies in infant mice showed that immune sera raised against either of the novel Hikojima vaccine strains protected baby mice against infection with virulent strains of both serotypes. This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole-cell vaccines.

  2. Genome-wide identification, phylogenetic and co-expression analysis of OsSET gene family in rice.

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    Zhanhua Lu

    Full Text Available BACKGROUND: SET domain is responsible for the catalytic activity of histone lysine methyltransferases (HKMTs during developmental process. Histone lysine methylation plays a crucial and diverse regulatory function in chromatin organization and genome function. Although several SET genes have been identified and characterized in plants, the understanding of OsSET gene family in rice is still very limited. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a systematic analysis was performed and revealed the presence of at least 43 SET genes in rice genome. Phylogenetic and structural analysis grouped SET proteins into five classes, and supposed that the domains out of SET domain were significant for the specific of histone lysine methylation, as well as the recognition of methylated histone lysine. Based on the global microarray, gene expression profile revealed that the transcripts of OsSET genes were accumulated differentially during vegetative and reproductive developmental stages and preferentially up or down-regulated in different tissues. Cis-elements identification, co-expression analysis and GO analysis of expression correlation of 12 OsSET genes suggested that OsSET genes might be involved in cell cycle regulation and feedback. CONCLUSIONS/SIGNIFICANCE: This study will facilitate further studies on OsSET family and provide useful clues for functional validation of OsSETs.

  3. The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter.

    Science.gov (United States)

    Darbani, Behrooz; Motawia, Mohammed Saddik; Olsen, Carl Erik; Nour-Eldin, Hussam H; Møller, Birger Lindberg; Rook, Fred

    2016-11-14

    Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters.

  4. Weighted gene co-expression network analysis of pneumocytes under exposure to a carcinogenic dose of chloroprene.

    Science.gov (United States)

    Guo, Yinghua; Xing, Yonghua

    2016-04-15

    Occupational exposure to chloroprene via inhalation may lead to acute toxicity and chronic pulmonary diseases, including lung cancer. Currently, most research is focused on epidemiological studies of chloroprene production workers. The specific molecular mechanism of carcinogenesis by chloroprene in lung tissues still remains obscure, and specific candidate therapeutic targets for lung cancer are lacking. The present study identifies specific gene modules and valuable hubs associated with lung cancer. We downloaded the dataset GSE40795 from the Gene Expression Omnibus (GEO) and divided the dataset into the non-carcinogenic dose chloroprene exposed mice group and the carcinogenic dose chloroprene exposed mice group. With a systemic biological view, we discovered significantly altered gene modules between the two groups and identified hub genes in the carcinogenic dose exposed group using weighted co-expression network analysis (WGCNA). A total of 2434 differentially expressed genes were identified. Twelve gene modules with multiple biological activities were related to the carcinogenesis of chloroprene in lung tissue. Seven hub genes that were critical for the carcinogenesis of chloroprene in lung tissue were ultimately identified, including Cftr, Hip1, Tbl1x, Ephx1, Cbr3, Antxr2 and Ccnd2. They were implicated in inflammatory response, cell transformation, gene transcription regulation, phase II detoxification, angiogenesis, cell adhesion, motility and the cell cycle. The seven hub genes may become valuable candidates for risk assessment biomarkers and therapeutic targets in lung cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. High co-expression of Sp1 and HER-2 is correlated with poor prognosis of gastric cancer patients.

    Science.gov (United States)

    Jiang, Weihua; Jin, Ziliang; Zhou, Fei; Cui, Jiujie; Wang, Lei; Wang, Liwei

    2015-09-01

    The aim of this study was to investigate co-expression of HER-2 and Sp1 in gastric cancer (GC) so as to determine whether these two proteins may be correlated with poor prognosis of GC patients. We examined the HER-2 overexpression and amplification and expression levels of Sp1 in 227 GC patients using immune-histochemical staining and fluorescence in situ hybridization. Data on clinicopathological features and relevant prognostic factors in these patients were analyzed. Of the 227 gastric cancer samples, 11.89% were positive for HER-2 overexpression/amplification under the new scoring system, and the frequency of negative, weak positive and strong positive expression of Sp1 was 14.98%, 48.01% and 37.0% respectively. No statistically positive correlation was observed between the expression levels of HER-2 and Sp1 in GC tissues. HER-2 overexpression was closely correlated to the Lauren type, degree of differentiation, tumor size and lymph node metastasis, and Sp1 as well (P HER-2 and Sp1 predicted poor survival in univariate analysis as well as in a Cox proportional hazards model. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean

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    Bingfu eGuo

    2015-10-01

    Full Text Available Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-PCR and Western blot revealed that target genes have been integrated into genome and expressed effectively at both mRNA and protein levels. Furthermore, the glyphosate tolerance analysis showed that no typical symptom was observed when compared with a glyphosate tolerant line HJ06-698 derived from GR1 transgenic soybean even at four-fold labeled rate of Roundup. Chlorophyll and shikimic acid content analysis of transgenic plant also revealed that these two indexes were not significantly altered after glyphosate application. These results indicated that co-expression of G2-EPSPS and GAT conferred high tolerance to the herbicide glyphosate in soybean. Therefore, combination of tolerant and degraded genes provides a new strategy for developing glyphosate tolerant transgenic crops.

  7. VSNL1 Co-expression networks in aging include calcium signaling, synaptic plasticity, and Alzheimer’s disease pathways

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    C W Lin

    2015-03-01

    Full Text Available The Visinin-like 1 (VSNL1 gene encodes Visinin-like protein 1, a peripheral biomarker for Alzheimer disease (AD. Little is known, however, about normal VSNL1 expression in brain and the biologic networks in which it participates. Frontal cortex gray matter from 209 subjects without neurodegenerative or psychiatric illness, ranging in age from 16–91, were processed on Affymetrix GeneChip 1.1 ST and Human SNP Array 6.0. VSNL1 expression was unaffected by age and sex, and not significantly associated with SNPs in cis or trans. VSNL1 was significantly co-expressed with genes in pathways for Calcium Signaling, AD, Long Term Potentiation, Long Term Depression, and Trafficking of AMPA Receptors. The association with AD was driven, in part, by correlation with amyloid precursor protein (APP expression. These findings provide an unbiased link between VSNL1 and molecular mechanisms of AD, including pathways implicated in synaptic pathology in AD. Whether APP may drive increased VSNL1 expression, VSNL1 drives increased APP expression, or both are downstream of common pathogenic regulators will need to be evaluated in model systems.

  8. The order of expression is a key factor in the production of active transglutaminase in Escherichia coli by co-expression with its pro-peptide

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    Liu Song

    2011-12-01

    Full Text Available Abstract Background Streptomyces transglutaminase (TGase is naturally synthesized as zymogen (pro-TGase, which is then processed to produce active enzyme by the removal of its N-terminal pro-peptide. This pro-peptide is found to be essential for overexpression of soluble TGase in E. coli. However, expression of pro-TGase by E. coli requires protease-mediated activation in vitro. In this study, we developed a novel co- expression method for the direct production of active TGase in E. coli. Results A TGase from S. hygroscopicus was expressed in E. coli only after fusing with the pelB signal peptide, but fusion with the signal peptide induced insoluble enzyme. Therefore, alternative protocol was designed by co-expressing the TGase and its pro-peptide as independent polypeptides under a single T7 promoter using vector pET-22b(+. Although the pro-peptide was co-expressed, the TGase fused without the signal peptide was undetectable in both soluble and insoluble fractions of the recombinant cells. Similarly, when both genes were expressed in the order of the TGase and the pro-peptide, the solubility of TGase fused with the signal peptide was not improved by the co-expression with its pro-peptide. Interestingly, active TGase was only produced by the cells in which the pro-peptide and the TGase were fused with the signal peptide and sequentially expressed. The purified recombinant and native TGase shared the similar catalytic properties. Conclusions Our results indicated that the pro-peptide can assist correct folding of the TGase inter-molecularly in E. coli, and expression of pro-peptide prior to that of TGase was essential for the production of active TGase. The co-expression strategy based on optimizing the order of gene expression could be useful for the expression of other functional proteins that are synthesized as a precursor.

  9. Pharmacological rescue of hERG currents carried out by G604S and wide type hERG co-expression.

    Science.gov (United States)

    Huo, Jianhua; Zhang, Aifeng; Guo, Xueyan; Qiang, Hua; Liu, Ping; Bai, Ling; Ma, Aiqun

    2016-09-01

    Mutations in human ether-a-go-go-related gene (hERG) can lead to type 2 long-QT syndrome (LQT2). The authors previously identified the hERG mutation G604S results in a loss of function and obviously decreased current amplitude and impaired channel protein trafficking when co-expressed with WT-hERG. The present study further investigates the biological and electrophysiological consequences of pharmacologic chaperones in HEK293 cells expressing G604S-hERG or co-expressing G604S-hERG and WT-hERG. It was found that a low temperature (27°C), thapsigargin, NS1643 and E-4031 fail to rescue the G604S mutation. Interestingly, only E-4031 treatment resulted in a significant increase in hERG currents in cells co-expressing G604S-hERG and WT-hERG, correspondingly more mature protein band at 155 kDa by Western blotting and an increased membrane staining by confocal microscopy. In addition, E-4031 treatment shifted the steady-state half maximal activation voltage (V1/2 ) of the inactivation curve by +8 mV in cells co-expressing G604S-hERG and WT-hERG. The present experimental results suggest that a G604S mutation is resistant to pharmacological rescue. E-4031 treatment resulted in a significant increase in hERG currents by promoting the hERG channel processing and trafficking in cells co-expressing G604S-hERG and WT-hERG. © 2016 John Wiley & Sons Australia, Ltd.

  10. Identification of estrogen receptor dimer selective ligands reveals growth-inhibitory effects on cells that co-express ERα and ERβ.

    Directory of Open Access Journals (Sweden)

    Emily Powell

    Full Text Available Estrogens play essential roles in the progression of mammary and prostatic diseases. The transcriptional effects of estrogens are transduced by two estrogen receptors, ERα and ERβ, which elicit opposing roles in regulating proliferation: ERα is proliferative while ERβ is anti-proliferative. Exogenous expression of ERβ in ERα-positive cancer cell lines inhibits cell proliferation in response to estrogen and reduces xenografted tumor growth in vivo, suggesting that ERβ might oppose ERα's proliferative effects via formation of ERα/β heterodimers. Despite biochemical and cellular evidence of ERα/β heterodimer formation in cells co-expressing both receptors, the biological roles of the ERα/β heterodimer remain to be elucidated. Here we report the identification of two phytoestrogens that selectively activate ERα/β heterodimers at specific concentrations using a cell-based, two-step high throughput small molecule screen for ER transcriptional activity and ER dimer selectivity. Using ERα/β heterodimer-selective ligands at defined concentrations, we demonstrate that ERα/β heterodimers are growth inhibitory in breast and prostate cells which co-express the two ER isoforms. Furthermore, using Automated Quantitative Analysis (AQUA to examine nuclear expression of ERα and ERβ in human breast tissue microarrays, we demonstrate that ERα and ERβ are co-expressed in the same cells in breast tumors. The co-expression of ERα and ERβ in the same cells supports the possibility of ERα/β heterodimer formation at physio- and pathological conditions, further suggesting that targeting ERα/β heterodimers might be a novel therapeutic approach to the treatment of cancers which co-express ERα and ERβ.

  11. Effects of a mutation in the HSPE1 gene encoding the mitochondrial co-chaperonin HSP10 and its potential association with a neurological and developmental disorder

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    Anne Sigaard Bie

    2016-10-01

    Full Text Available We here report molecular investigations of a missense mutation in the HSPE1 gene encoding the HSP10 subunit of the HSP60/ HSP10 chaperonin complex that assists protein folding in the mitochondrial matrix. The mutation was identified in an infant who came to clinical attention due to infantile spasms at three months of age. Clinical exome sequencing revealed heterozygosity for a HSPE1 NM_002157.2:c.217C>T de novo mutation causing replacement of leucine with phenylalanine at position 73 of the HSP10 protein. This variation has never been observed in public exome sequencing databases or the literature.To evaluate whether the mutation may be disease-associated we investigated its effects by in vitro and ex vivo studies. Our in vitro studies indicated that the purified mutant protein was functional, yet its thermal stability, spontaneous refolding propensity, and resistance to proteolytic treatment were profoundly impaired. Mass spectrometric analysis of patient fibroblasts revealed barely detectable levels of HSP10-p.Leu73Phe protein resulting in an almost 2-fold decrease of the ratio of HSP10 to HSP60 subunits. Amounts of the mitochondrial superoxide dismutase SOD2, a protein whose folding is known to strongly depend on the HSP60/HSP10 complex, were decreased to approximately 20% in patient fibroblasts in spite of unchanged SOD2 transcript levels. As a likely consequence, mitochondrial superoxide levels were increased about 2-fold. Although we cannot exclude other causative or contributing factors, our experimental data support the notion that the HSP10-p.Leu73Phe mutation could be the cause or a strong contributing factor for the disorder in the described patient.

  12. Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

    Science.gov (United States)

    Marchenko, N Iu; Marchenkov, V V; Kaĭsheva, A L; Kashparov, I A; Kotova, N V; Kaliman, P A; Semisotnov, G V

    2006-12-01

    The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.

  13. Co-expression of VAL- and TMT-opsins uncovers ancient photosensory interneurons and motorneurons in the vertebrate brain.

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    Ruth M Fischer

    Full Text Available The functional principle of the vertebrate brain is often paralleled to a computer: information collected by dedicated devices is processed and integrated by interneuron circuits and leads to output. However, inter- and motorneurons present in today's vertebrate brains are thought to derive from neurons that combined sensory, integration, and motor function. Consistently, sensory inter-motorneurons have been found in the simple nerve nets of cnidarians, animals at the base of the evolutionary lineage. We show that light-sensory motorneurons and light-sensory interneurons are also present in the brains of vertebrates, challenging the paradigm that information processing and output circuitry in the central brain is shielded from direct environmental influences. We investigated two groups of nonvisual photopigments, VAL- and TMT-Opsins, in zebrafish and medaka fish; two teleost species from distinct habitats separated by over 300 million years of evolution. TMT-Opsin subclasses are specifically expressed not only in hypothalamic and thalamic deep brain photoreceptors, but also in interneurons and motorneurons with no known photoreceptive function, such as the typeXIV interneurons of the fish optic tectum. We further show that TMT-Opsins and Encephalopsin render neuronal cells light-sensitive. TMT-Opsins preferentially respond to blue light relative to rhodopsin, with subclass-specific response kinetics. We discovered that tmt-opsins co-express with val-opsins, known green light receptors, in distinct inter- and motorneurons. Finally, we show by electrophysiological recordings on isolated adult tectal slices that interneurons in the position of typeXIV neurons respond to light. Our work supports "sensory-inter-motorneurons" as ancient units for brain evolution. It also reveals that vertebrate inter- and motorneurons are endowed with an evolutionarily ancient, complex light-sensory ability that could be used to detect changes in ambient light spectra

  14. Mining Temporal Protein Complex Based on the Dynamic PIN Weighted with Connected Affinity and Gene Co-Expression.

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    Xianjun Shen

    Full Text Available The identification of temporal protein complexes would make great contribution to our knowledge of the dynamic organization characteristics in protein interaction networks (PINs. Recent studies have focused on integrating gene expression data into static PIN to construct dynamic PIN which reveals the dynamic evolutionary procedure of protein interactions, but they fail in practice for recognizing the active time points of proteins with low or high expression levels. We construct a Time-Evolving PIN (TEPIN with a novel method called Deviation Degree, which is designed to identify the active time points of proteins based on the deviation degree of their own expression values. Owing to the differences between protein interactions, moreover, we weight TEPIN with connected affinity and gene co-expression to quantify the degree of these interactions. To validate the efficiencies of our methods, ClusterONE, CAMSE and MCL algorithms are applied on the TEPIN, DPIN (a dynamic PIN constructed with state-of-the-art three-sigma method and SPIN (the original static PIN to detect temporal protein complexes. Each algorithm on our TEPIN outperforms that on other networks in terms of match degree, sensitivity, specificity, F-measure and function enrichment etc. In conclusion, our Deviation Degree method successfully eliminates the disadvantages which exist in the previous state-of-the-art dynamic PIN construction methods. Moreover, the biological nature of protein interactions can be well described in our weighted network. Weighted TEPIN is a useful approach for detecting temporal protein complexes and revealing the dynamic protein assembly process for cellular organization.

  15. Enhanced resistance to Sclerotinia sclerotiorum in Brassica napus by co-expression of defensin and chimeric chitinase genes.

    Science.gov (United States)

    Zarinpanjeh, Nasim; Motallebi, Mostafa; Zamani, Mohammad Reza; Ziaei, Mahboobeh

    2016-11-01

    Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the major fungal diseases of Brassica napus L. To develop resistance against this fungal disease, the defensin gene from Raphanus sativus and chimeric chit42 from Trichoderma atroviride with a C-terminal fused chitin-binding domain from Serratia marcescens were co-expressed in canola via Agrobacterium-mediated transformation. Twenty transformants were confirmed to carry the two transgenes as detected by polymerase chain reaction (PCR), with 4.8 % transformation efficiency. The chitinase activity of PCR-positive transgenic plants were measured in the presence of colloidal chitin, and five transgenic lines showing the highest chitinase activity were selected for checking the copy number of the transgenes through Southern blot hybridisation. Two plants carried a single copy of the transgenes, while the remainder carried either two or three copies of the transgenes. The antifungal activity of two transgenic lines that carried a single copy of the transgenes (T4 and T10) was studied by a radial diffusion assay. It was observed that the constitutive expression of these transgenes in the T4 and T10 transgenic lines suppressed the growth of S. sclerotiorum by 49 % and 47 %, respectively. The two transgenic lines were then let to self-pollinate to produce the T2 generation. Greenhouse bioassays were performed on the transgenic T2 young leaves by challenging with S. sclerotiorum and the results revealed that the expression of defensin and chimeric chitinase from a heterologous source in canola demonstrated enhanced resistance against sclerotinia stem rot disease.

  16. Constitutive activity of the cannabinoid CB1 receptor regulates the function of co-expressed Mu opioid receptors.

    Science.gov (United States)

    Canals, Meritxell; Milligan, Graeme

    2008-04-25

    The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.

  17. Mining Temporal Protein Complex Based on the Dynamic PIN Weighted with Connected Affinity and Gene Co-Expression.

    Science.gov (United States)

    Shen, Xianjun; Yi, Li; Jiang, Xingpeng; He, Tingting; Hu, Xiaohua; Yang, Jincai

    2016-01-01

    The identification of temporal protein complexes would make great contribution to our knowledge of the dynamic organization characteristics in protein interaction networks (PINs). Recent studies have focused on integrating gene expression data into static PIN to construct dynamic PIN which reveals the dynamic evolutionary procedure of protein interactions, but they fail in practice for recognizing the active time points of proteins with low or high expression levels. We construct a Time-Evolving PIN (TEPIN) with a novel method called Deviation Degree, which is designed to identify the active time points of proteins based on the deviation degree of their own expression values. Owing to the differences between protein interactions, moreover, we weight TEPIN with connected affinity and gene co-expression to quantify the degree of these interactions. To validate the efficiencies of our methods, ClusterONE, CAMSE and MCL algorithms are applied on the TEPIN, DPIN (a dynamic PIN constructed with state-of-the-art three-sigma method) and SPIN (the original static PIN) to detect temporal protein complexes. Each algorithm on our TEPIN outperforms that on other networks in terms of match degree, sensitivity, specificity, F-measure and function enrichment etc. In conclusion, our Deviation Degree method successfully eliminates the disadvantages which exist in the previous state-of-the-art dynamic PIN construction methods. Moreover, the biological nature of protein interactions can be well described in our weighted network. Weighted TEPIN is a useful approach for detecting temporal protein complexes and revealing the dynamic protein assembly process for cellular organization.

  18. Gene co-expression analyses differentiate networks associated with diverse cancers harbouring TP53 missense or null mutations

    Directory of Open Access Journals (Sweden)

    Kathleen Oros Klein

    2016-08-01

    Full Text Available In a variety of solid cancers, missense mutations in the well-established TP53 tumour suppressor gene may lead to presence of a partially-functioning protein molecule, whereas mutations affecting the protein encoding reading frame, often referred to as null mutations, result in the absence of p53 protein. Both types of mutations have been observed in the same cancer type. As the resulting tumour biology may be quite different between these two groups, we used RNA-sequencing data from The Cancer Genome Atlas (TCGA from four different cancers with poor prognosis, namely ovarian, breast, lung and skin cancers, to compare the patterns of co-expression of genes in tumours grouped according to their TP53 missense or null mutation status. We used Weighted Gene Coexpression Network analysis (WGCNA and a new test statistic built on differences between groups in the measures of gene connectivity. For each cancer, our analysis identified a set of genes showing differential coexpression patterns between the TP53 missense- and null mutation-carrying groups that was robust to the choice of the tuning parameter in WGCNA. After comparing these sets of genes across the four cancers, one gene (KIR3DL2 consistently showed differential coexpression patterns between the null and missense groups. KIR3DL2 is known to play an important role in regulating the immune response, which is consistent with our observation that this gene’s strongly-correlated partners implicated many immune-related pathways. Examining mutation-type-related changes in correlations between sets of genes may provide new insight into tumour biology.

  19. Production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expressing bacterial PNGase F.

    Science.gov (United States)

    Mamedov, Tarlan; Ghosh, Ananya; Jones, R Mark; Mett, Vadim; Farrance, Christine E; Musiychuk, Konstantin; Horsey, April; Yusibov, Vidadi

    2012-09-01

    Application of tools of molecular biology and genomics is increasingly leading towards the development of recombinant protein-based biologics. As such, it is leading to an increased diversity of targets that have important health applications and require more flexible approaches for expression because of complex post-translational modifications. For example, Plasmodium parasites may have complex post-translationally modified proteins such as Pfs48/45 that do not carry N-linked glycans (Exp. Parasitol. 1998; 90, 165.) but contain potential N-linked glycosylation sites that can be aberrantly glycosylated during expression in mammalian and plant systems. Therefore, it is important to develop strategies for producing non-glycosylated forms of these targets to preserve biological activity and native conformation. In this study, we are describing in vivo deglycosylation of recombinant N-glycosylated proteins as a result of their transient co-expression with bacterial PNGase F (Peptide: N-glycosidase F). In addition, we show that the recognition of an in vivo deglycosylated plant-produced malaria vaccine candidate, Pfs48F1, by monoclonal antibodies I, III and V raised against various epitopes (I, III and V) of native Pfs48/45 of Plasmodium falciparum, was significantly stronger compared to that of the glycosylated form of plant-produced Pfs48F1. To our knowledge, neither in vivo enzymatic protein deglycosylation has been previously achieved in any eukaryotic system, including plants, nor has bacterial PNGase F been expressed in the plant system. Thus, here, we report for the first time the expression in plants of an active bacterial enzyme PNGase F and the production of recombinant proteins of interest in a non-glycosylated form. © 2012 Fraunhofer USA Center for Molecular Biotechnology. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  20. Electron transfer in a human inducible nitric oxide synthase oxygenase/FMN construct co-expressed with the N-terminal globular domain of calmodulin

    Science.gov (United States)

    Fan, Weihong; Dupont, Andrea; Guillemette, J. Guy; Ghosh, Dipak K.

    2010-01-01

    The FMN–heme intraprotein electron transfer (IET) kinetics in a human iNOS oxygenase/FMN (oxyFMN) construct co-expressed with NCaM, a calmodulin (CaM) construct that includes only its N-terminal globular domain, were determined by laser flash photolysis. The IET rate constant is significantly decreased by nearly 4-fold (compared to the iNOS oxyFMN co-expressed with full length CaM). This supports an important role of full length CaM in proper interdomain FMN/heme alignment in iNOS. The IET process was not observed with added excess EDTA, suggesting that Ca2+ depletion results in the FMN domain moving away from the heme domain. The results indicate that a Ca2+-dependent reorganization of the NCaM construct could cause a major modification of the NCaM/iNOS association resulting in a loss of IET. PMID:20868689

  1. Production of d-allulose from d-glucose by Escherichia coli transformant cells co-expressing d-glucose isomerase and d-psicose 3-epimerase genes.

    Science.gov (United States)

    Zhang, Wenli; Li, Hao; Jiang, Bo; Zhang, Tao; Mu, Wanmeng

    2017-08-01

    d-Allulose is a novel and low-calorie rare monosaccharide that is a C-3 epimer of d-fructose. Because of its excellent physiological properties and commercial potential, d-allulose has attracted researchers' interests. Based on the Izumoring strategy, d-allulose is converted from d-fructose by d-psicose 3-epimerase (DPEase), while d-fructose is converted from d-glucose by d-glucose isomerase (GIase). In this study, we created a cellular system capable of converting d-glucose to d-allulose in a one-step process that co-expressed the GIase from Acidothermus cellulolyticus and the DPEase from Dorea sp. CAG. The co-expression plasmid pETDuet-Dosp-DPE/Acce-GI was generated and transformed into Escherichia coli BL21(DE3) cells. The recombinant co-expression cells exhibited maximum catalytic activity at pH 6.5 and 75 °C. These cells were thermostable at less than 60 °C. The addition of Co 2+ significantly increased the catalytic activity by 10.8-fold. When the reaction equilibrium was reached, the ratio of d-glucose, d-fructose and d-allulose was approximately 6.5:7:3, respectively. A recombinant co-expression strain that catalysed the bioconversion of d-allulose from d-glucose in a one-step process was created and characterised. When adding 500 g L -1 d-glucose as a substrate, 204.3 g L -1 d-fructose and 89.1 g L -1 d-allulose were produced. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  2. The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter

    OpenAIRE

    Behrooz Darbani; Mohammed Saddik Motawia; Carl Erik Olsen; Nour-Eldin, Hussam H.; Birger Lindberg Møller; Fred Rook

    2016-01-01

    Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expresse...

  3. Co-Expression of HER Family Members in Patients with Dukes’ C and D Colon Cancer and Their Impacts on Patient Prognosis and Survival

    Science.gov (United States)

    Khelwatty, Said Abdullah; Essapen, Sharadah; Bagwan, Izhar; Green, Margaret; Seddon, Alan Michael; Modjtahedi, Helmout

    2014-01-01

    The human epidermal growth factor receptor (EGFR) is an important therapeutic target in patients with metastatic colorectal cancer and anti-EGFR antibodies cetuximab and panitumumab have been approved for the treatment of such patients. Despite these advances, the duration of response in some patients can be limited. Since, EGFR is capable of forming heterodimers with the other members of the HER (Human epidermal receptor) family, it is important to investigate the co-expression and prognostic significance of all members of the HER family in colorectal cancer patients. The expression of the HER family members were determined in tumour specimens from 86 patients with Dukes’ C and D (metastatic) colon cancer using immunohistochemistry. Sections were scored by the percentage of positive tumour cells and intensity of staining. Their associations with clinicopathological parameters, and overall survival and disease free survival were evaluated using univariate and multivariate analysis. Overall, 43%, 77%, 52% and 92% of the cases were EGFR, HER-2, HER-3 and HER-4 positive respectively. Interestingly, 35%, 24%, 43%, and 18% of the cases had co-expression of EGFR/HER-2, EGFR/HER-3, EGFR/HER-4 and all four members of the HER family respectively. Of these, only the expression of EGFR and co-expression of EGFR/HER-4 were associated with poorer disease-free survival in both univariate and multivariate analysis. Co-expression of all members of the HER family in colon cancer supports the need for further investigations on their predictive value for response to therapy with anti-EGFR mAbs and whether such sub-population of patients may benefit from therapy with the new generation of pan-HER inhibitors. PMID:24609222

  4. Co-expression of tonoplast Cation/H(+) antiporter and H(+)-pyrophosphatase from xerophyte Zygophyllum xanthoxylum improves alfalfa plant growth under salinity, drought and field conditions.

    Science.gov (United States)

    Bao, Ai-Ke; Du, Bao-Qiang; Touil, Leila; Kang, Peng; Wang, Qiang-Long; Wang, Suo-Min

    2016-03-01

    Salinity and drought are major environmental factors limiting the growth and productivity of alfalfa worldwide as this economically important legume forage is sensitive to these kinds of abiotic stress. In this study, transgenic alfalfa lines expressing both tonoplast NXH and H(+)-PPase genes, ZxNHX and ZxVP1-1 from the xerophyte Zygophyllum xanthoxylum L., were produced via Agrobacterium tumefaciens-mediated transformation. Compared with wild-type (WT) plants, transgenic alfalfa plants co-expressing ZxNHX and ZxVP1-1 grew better with greater plant height and dry mass under normal or stress conditions (NaCl or water-deficit) in the greenhouse. The growth performance of transgenic alfalfa plants was associated with more Na(+), K(+) and Ca(2+) accumulation in leaves and roots, as a result of co-expression of ZxNHX and ZxVP1-1. Cation accumulation contributed to maintaining intracellular ions homoeostasis and osmoregulation of plants and thus conferred higher leaf relative water content and greater photosynthesis capacity in transgenic plants compared to WT when subjected to NaCl or water-deficit stress. Furthermore, the transgenic alfalfa co-expressing ZxNHX and ZxVP1-1 also grew faster than WT plants under field conditions, and most importantly, exhibited enhanced photosynthesis capacity by maintaining higher net photosynthetic rate, stomatal conductance, and water-use efficiency than WT plants. Our results indicate that co-expression of tonoplast NHX and H(+)-PPase genes from a xerophyte significantly improved the growth of alfalfa, and enhanced its tolerance to high salinity and drought. This study laid a solid basis for reclaiming and restoring saline and arid marginal lands as well as improving forage yield in northern China. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  5. STARNET 2: a web-based tool for accelerating discovery of gene regulatory networks using microarray co-expression data

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    VanBuren Vincent

    2009-10-01

    Full Text Available Abstract Background Although expression microarrays have become a standard tool used by biologists, analysis of data produced by microarray experiments may still present challenges. Comparison of data from different platforms, organisms, and labs may involve complicated data processing, and inferring relationships between genes remains difficult. Results STARNET 2 is a new web-based tool that allows post hoc visual analysis of correlations that are derived from expression microarray data. STARNET 2 facilitates user discovery of putative gene regulatory networks in a variety of species (human, rat, mouse, chicken, zebrafish, Drosophila, C. elegans, S. cerevisiae, Arabidopsis and rice by graphing networks of genes that are closely co-expressed across a large heterogeneous set of preselected microarray experiments. For each of the represented organisms, raw microarray data were retrieved from NCBI's Gene Expression Omnibus for a selected Affymetrix platform. All pairwise Pearson correlation coefficients were computed for expression profiles measured on each platform, respectively. These precompiled results were stored in a MySQL database, and supplemented by additional data retrieved from NCBI. A web-based tool allows user-specified queries of the database, centered at a gene of interest. The result of a query includes graphs of correlation networks, graphs of known interactions involving genes and gene products that are present in the correlation networks, and initial statistical analyses. Two analyses may be performed in parallel to compare networks, which is facilitated by the new HEATSEEKER module. Conclusion STARNET 2 is a useful tool for developing new hypotheses about regulatory relationships between genes and gene products, and has coverage for 10 species. Interpretation of the correlation networks is supported with a database of previously documented interactions, a test for enrichment of Gene Ontology terms, and heat maps of correlation

  6. Co-expression and promoter content analyses assign a role in biotic and abiotic stress responses to plant natriuretic peptides

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    Murray Shane

    2008-02-01

    Full Text Available Abstract Background Plant natriuretic peptides (PNPs are a class of systemically mobile molecules distantly related to expansins. While several physiological responses to PNPs have been reported, their biological role has remained elusive. Here we use a combination of expression correlation analysis, meta-analysis of gene expression profiles in response to specific stimuli and in selected mutants, and promoter content analysis to infer the biological role of the Arabidopsis thaliana PNP, AtPNP-A. Results A gene ontology analysis of AtPNP-A and the 25 most expression correlated genes revealed a significant over representation of genes annotated as part of the systemic acquired resistance (SAR pathway. Transcription of these genes is strongly induced in response to salicylic acid (SA and its functional synthetic analogue benzothiadiazole S-methylester (BTH, a number of biotic and abiotic stresses including many SA-mediated SAR-inducing conditions, as well as in the constitutive SAR expressing mutants cpr5 and mpk4 which have elevated SA levels. Furthermore, the expression of AtPNP-A was determined to be significantly correlated with the SAR annotated transcription factor, WRKY 70, and the promoters of AtPNP-A and the correlated genes contain an enrichment in the core WRKY binding W-box cis-elements. In constitutively expressing WRKY 70 lines the expression of AtPNP-A and the correlated genes, including the SAR marker genes, PR-2 and PR-5, were determined to be strongly induced. Conclusion The co-expression analyses, both in wild type and mutants, provides compelling evidence that suggests AtPNP-A may function as a component of plant defence responses and SAR in particular. The presented evidence also suggests that the expression of AtPNP-A is controlled by WRKY transcription factors and WRKY 70 in particular. AtPNP-A shares many characteristics with PR proteins in that its transcription is strongly induced in response to pathogen challenges, it

  7. Individual and co-expression patterns of nerve growth factor and heme oxygenase-1 predict shorter survival of gastric carcinoma patients.

    Science.gov (United States)

    Noh, Sang Jae; Kim, Kyoung Min; Jang, Kyu Yun

    2017-07-05

    Nerve growth factor (NGF) is a neurotrophic factor which regulates cell development and proliferation. Recently, it has been suggested that NGF induces heme oxygenase-1 (HO1) expression, and that both NGF and HO1 are involved in the progression of malignant human tumors. However, exact roles of NGF and HO1 in tumorigenesis remain controversial. Therefore, we investigated the expression and correlation of NGF and HO1 in human gastric carcinoma tissues. We examined immunohistochemical expression of NGF and HO1 in 167 gastric carcinomas and compared with various prognostic clinicopathological factors. The expression of NGF and HO1 was positive in 40% (67/167) and 51% (85/167) of cases, respectively, and their expression was significantly correlated with each other (p < 0.001). Individual expression patterns of NGF and HO1, and co-expression pattern of these two molecules were significantly associated with shorter survival by univariate analysis. HO1 expression (overall survival; p < 0.001, relapse-free survival; p = 0.002) and co-expression pattern of NGF and HO1 (overall survival; p = 0.002, relapse-free survival; p = 0.003) were independent poor prognostic indicators of gastric carcinoma patients by multivariate analysis. These results demonstrate that the individual and co-expression patterns of NGF and HO1 might be used as prognostic indicators for gastric carcinoma patients.

  8. Keratitis-Ichthyosis-Deafness syndrome-associated Cx26 mutants produce nonfunctional gap junctions but hyperactive hemichannels when co-expressed with wild type Cx43

    Science.gov (United States)

    García, Isaac E.; Maripillán, Jaime; Jara, Oscar; Ceriani, Ricardo; Palacios-Muñoz, Angelina; Ramachandran, Jayalakshimi; Olivero, Pablo; Pérez-Acle, Tomás; González, Carlos; Sáez, Juan C.; Contreras, Jorge E.; Martínez, Agustín D.

    2015-01-01

    Mutations in Cx26 gene are found in most cases of human genetic deafness. Some mutations produce syndromic deafness associated with skin disorders, like Keratitis Ichthyosis Deafness syndrome (KID). Because in the human skin Cx26 is co-expressed with other connexins, like Cx43 and Cx30, and since KID syndrome is inherited as autosomal dominant condition, it is possible that KID mutations change the way Cx26 interacts with other co-expressed connexins. Indeed, some Cx26 syndromic mutations showed gap junction dominant negative effect when co-expressed with wild type connexins, including Cx26 and Cx43. The nature of these interactions and the consequences on hemichannels and gap junction channels functions remain unknown. In this study we demonstrate that syndromic mutations at the N-terminus segment of Cx26, change connexin oligomerization compatibility, allowing aberrant interactions with Cx43. Strikingly, heteromeric oligomer formed by Cx43/Cx26 (syndromic mutants) show exacerbated hemichannel activity, but nonfunctional gap junction channels; this also occurs for those Cx26 KID mutants that do not show functional homomeric hemichannels. Heterologous expression of these hyperactive heteromeric hemichannels increases cell membrane permeability, favoring ATP release and Ca2+ overload. The functional paradox produced by oligomerization of Cx43 and Cx26 KID mutants could underlie the severe syndromic phenotype in human skin. PMID:25625422

  9. Production of d-psicose from d-glucose by co-expression of d-psicose 3-epimerase and xylose isomerase.

    Science.gov (United States)

    Chen, Xiaoyan; Wang, Wen; Xu, Jingliang; Yuan, Zhenhong; Yuan, Tao; Zhang, Yu; Liang, Cuiyi; He, Minchao; Guo, Ying

    2017-10-01

    d-Psicose has been drawing increasing attention in recent years because of its medical and health applications. The production of d-psicose from d-glucose requires the co-expression and synergistic action of xylose isomerase and d-psicose 3-epimerase. To co-express these genes, vector pET-28a(+)-dual containing two T7 promoters and RBS sites and an Multiple Cloning Sites was constructed using the Escherichia coli expression plasmid pET-28a(+). The xylose isomerase gene from E. coli MG1665 and the d-psicose 3-epimerase gene from Agrobacterium tumefaciens CGMCC 1.1488 were cloned and co-expressed in E. coli BL21(DE3). After 24h incubation with the dual enzyme system at 40°C, the sugar conversion ratio from d-glucose to d-psicose reached 10%. The optimal conditions were 50°C, pH 7.5 with Co2+ and Mg2+. The d-psicose yields from sugarcane bagasse and microalgae hydrolysate were 1.42 and 1.69g/L, respectively. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Impact of estrogen receptor (ER) and human epidermal growth factor receptor-2 (HER2) co-expression on breast cancer disease characteristics: implications for tumor biology and research.

    Science.gov (United States)

    Alqaisi, Abeer; Chen, Li; Romond, Edward; Chambers, Mara; Stevens, Mark; Pasley, Grace; Awasthi, Mukta; Massarweh, Suleiman

    2014-11-01

    ER and HER2 are critical drivers of breast cancer biology and can interact when co-expressed, but less data describe the impact of ER/HER2 co-expression on clinical disease characteristics. We studied the impact of ER and HER2 (co)-expression in a cohort of 1,187 patients with invasive breast cancer and compared disease characteristics among different groups according to ER and HER2 status. Age, tumor size, grade, nodal status, TNM stage, and metastatic sites were compared and significance determined using the appropriate t tests. All p values were two-tailed. Compared to ER-negative/HER2-negative disease as the control group, ER expression was associated with older age, smaller tumors, lower grade, earlier TNM stage, and increased bone involvement in de novo metastasis, while HER2 had no significant impact on these characteristics. ER and HER2 co-expression was associated with lower grade and higher bone involvement in de novo metastasis, reflecting a retained impact for ER. HER2 impact on ER-positive disease was reflected by younger age, higher grade and TNM stage, and increased frequency of visceral involvement in de novo metastasis. Within the ER-positive/HER2-positive group, triple positive breast cancer (ER+/PgR+/HER2+) was associated with younger age compared to ER+/PgR-/HER2+ disease (mean age of 50.8 vs. 56 years, p = 0.0226). PgR was also associated with younger age in ER+/HER2- disease with a mean age of 57.6 years in ER+/PgR+/HER2- disease vs. 63.4 years in ER+/PgR-/HER2- disease (p impact on breast cancer characteristics, including a retained impact when co-expressed with HER2. Similarly, HER2 dramatically modulates ER-positive breast cancer making it more aggressive. PgR association with young age may be related to hormonal levels of the premenopausal state, with HER2 providing an earlier growth advantage in triple positive disease, suggesting a specific dependence for this subset on high estrogen levels.

  11. A DNA vaccine co-expressing Trichinella spiralis MIF and MCD-1 with murine ubiquitin induces partial protective immunity in mice.

    Science.gov (United States)

    Tang, F; Xu, L; Yan, R; Song, X; Li, X

    2013-03-01

    Co-expression of Trichinella spiralis macrophage migration inhibitory factor (TsMIF) with T. spiralis cystatin-like domain protein (TsMCD-1) in a DNA vaccine induces a Th1 immune response and partial protection against T. spiralis infection. The present study evaluated whether co-expression of mouse ubiquitin (Ub) with TsMIF and TsMCD-1 might improve the immune response against T. spiralis infection. Groups of BALB/c mice were immunized twice at 2-week intervals with 100 μg of plasmid DNA encoding either a TsMIF-TsMCD-1 fusion protein (pVAX1-Tsmif-Tsmcd-1) or an Ub-co-expressing triple fusion protein Ub-TsMIF-TsMCD-1 (pVAX1-Ub-Tsmif-Tsmcd-1). Control animals were immunized with pVAX1-Ub or blank vector plasmid. Specific antibody levels (IgG, IgG1, IgG2a, IgG2b, IgM, IgA, IgE) against the recombinant protein TsMIF-TsMCD-1, serum cytokines (interferon (IFN)-γ, interleukin (IL)-4, IL-5, transforming growth factor (TGF)-β1 and IL-17), CD4+/CD8+ T cells and cytotoxic T lymphocyte (CTL) responses were monitored. Challenge infection was performed 2 weeks after the second immunization and worm burden was assayed at 35 days post-challenge. Antibody responses induced by pVAX1-Ub-Tsmif-Tsmcd-1 were significantly lower than for TsMIF-TsMCD-1, but the vaccine induced increased levels of Th1 cytokine (IFN-γ) and increased T-cell cytotoxicity. The reduction of worm burden (37.95%) following immunization with pVAX1-Ub-Tsmif-Tsmcd-1 was significantly greater than that induced by the pVAX1-Tsmif-Tsmcd-1 vaccine (23.17%; P< 0.05).

  12. Co-expression of anti-rotavirus proteins (llama VHH antibody fragments in Lactobacillus: development and functionality of vectors containing two expression cassettes in tandem.

    Directory of Open Access Journals (Sweden)

    Gökçe Günaydın

    Full Text Available Rotavirus is an important pediatric pathogen, causing severe diarrhea and being associated with a high mortality rate causing approximately 500 000 deaths annually worldwide. Even though some vaccines are currently available, their efficacy is lower in the developing world, as compared to developed countries. Therefore, alternative or complementary treatment options are needed in the developing countries where the disease burden is the largest. The effect of Lactobacillus in promoting health and its use as a vehicle for delivery of protein and antibody fragments was previously shown. In this study, we have developed co-expression vectors enabling Lactobacillus paracasei BL23 to produce two VHH fragments against rotavirus (referred to as anti-rotavirus proteins 1 and 3, ARP1 and ARP3 as secreted and/or surface displayed products. ARP1 and ARP3 fragments were successfully co-expressed as shown by Western blot and flow cytometry. In addition, engineered Lactobacillus produced VHH antibody fragments were shown to bind to a broad range of rotavirus serotypes (including the human rotavirus strains 69M, Va70, F45, DS1, Wa and ST3 and simian rotavirus strains including RRV and SA11, by flow cytometry and ELISA. Hereby, we have demonstrated for the first time that when RRV was captured by one VHH displayed on the surface of co-expressor Lactobacillus, targeting other epitope was possible with another VHH secreted from the same bacterium. Therefore, Lactobacillus producing two VHH antibody fragments may potentially serve as treatment against rotavirus with a reduced risk of development of escape mutants. This co-expression and delivery platform can also be used for delivery of VHH fragments against a variety of mucosal pathogens or production of other therapeutic molecules.

  13. Co-expression and synergism analysis of Vip3Aa29 and Cyt2Aa3 insecticidal proteins from Bacillus thuringiensis.

    Science.gov (United States)

    Yu, Xiumei; Liu, Tao; Sun, Zhiguang; Guan, Peng; Zhu, Jun; Wang, Shiquan; Li, Shuangcheng; Deng, Qiming; Wang, Lingxia; Zheng, Aiping; Li, Ping

    2012-04-01

    Vegetative insecticidal protein (Vip3) from Bacillus thuringiensis shows high activity against lepidopteran insects. Cytolytic δ-endotoxin (Cyt) also has high toxicity to dipteran larvae and synergism with other crystal proteins (Cry), but synergism between Cyt and Vip3 proteins has not been tested. We analyzed for synergism between Cyt2Aa3 and Vip3Aa29. Both cyt2Aa3 and vip3Aa29 genes were co-expressed in Escherichia coli strain BL21 carried on vector pCOLADuet-1. Vip3Aa29 showed insecticidal activity against Chilo suppressalis and Spodoptera exigua, with 50% lethal concentration (LC(50)) at 24.0 and 36.6 μg ml(-1), respectively. It could also inhibit Helicoverpa armigera growth, with 50% inhibition concentration at 22.6 μg ml(-1). While Cyt2Aa3 was toxic to Culex quinquefasciatus (LC(50): 0.53 μg ml(-1)) and Chironomus tepperi (LC(50): 36 μg ml(-1)), it did not inhibit C. suppressalis, S. exigua, and H. armigera. However, the co-expression of Cyt2Aa3 and Vip3Aa29 showed synergistic effect on C. suppressalis and S. exigua, and the individual activities were strengthened 3.35- and 4.34-fold, respectively. The co-expression had no synergism against C. tepperi and H. armigera, but exerted some antagonistic effect on Cx. quinquefasciatus. The synergism between Cyt2Aa and Vip3Aa was thus discovered for the first time, which confirmed that Cyt toxin can enhance the toxicity of other toxins against some non-target insects. By synergism analysis, the effectiveness of microbial insecticides can be verified.

  14. Mct8 and trh co-expression throughout the hypothalamic paraventricular nucleus is modified by dehydration-induced anorexia in rats.

    Science.gov (United States)

    Alvarez-Salas, Elena; Mengod, Guadalupe; García-Luna, Cinthia; Soberanes-Chávez, Paulina; Matamoros-Trejo, Gilberto; de Gortari, Patricia

    2016-04-01

    Thyrotropin-releasing hormone (TRH) is a neuropeptide with endocrine and neuromodulatory effects. TRH from the paraventricular hypothalamic nucleus (PVN) participates in the control of energy homeostasis; as a neuromodulator TRH has anorexigenic effects. Negative energy balance decreases PVN TRH expression and TSH concentration; in contrast, a particular model of anorexia (dehydration) induces in rats a paradoxical increase in TRH expression in hypophysiotropic cells from caudal PVN and high TSH serum levels, despite their apparent hypothalamic hyperthyroidism and low body weight. We compared here the mRNA co-expression pattern of one of the brain thyroid hormones' transporters, the monocarboxylate transporter-8 (MCT8) with that of TRH in PVN subdivisions of dehydration-induced anorexic (DIA) and control rats. Our aim was to identify whether a low MCT8 expression in anorexic rats could contribute to their high TRH mRNA content.We registered daily food intake and body weight of 7-day DIA and control rats and analyzed TRH and MCT8 mRNA co-expression throughout the PVN by double in situ hybridization assays. We found that DIA rats showed increased number of TRHergic cells in caudal PVN, as well as a decreased percentage of TRH-expressing neurons that co-expressed MCT8 mRNA signal. Results suggest that the reduced proportion of double TRH/MCT8 expressing cells may be limiting the entry of hypothalamic triiodothyronine to the greater number of TRH-expressing neurons from caudal PVN and be in part responsible for the high TRH expression in anorexia rats and for the lack of adaptation of their hypothalamic-pituitary-thyroid axis to their low food intake.

  15. Transient Co-Expression of Post-Transcriptional Gene Silencing Suppressors for Increased in Planta Expression of a Recombinant Anthrax Receptor Fusion Protein

    Directory of Open Access Journals (Sweden)

    Kittipong Rattanaporn

    2011-08-01

    Full Text Available Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin, CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA domain of human capillary morphogenesis 2 (CMG2, an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG. We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS: p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI, with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI.

  16. Transient co-expression of post-transcriptional gene silencing suppressors for increased in planta expression of a recombinant anthrax receptor fusion protein.

    Science.gov (United States)

    Arzola, Lucas; Chen, Junxing; Rattanaporn, Kittipong; Maclean, James M; McDonald, Karen A

    2011-01-01

    Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS): p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI), with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI.

  17. Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase

    Directory of Open Access Journals (Sweden)

    Pappu Kameshwari M

    2011-06-01

    Full Text Available Abstract Background Collagens require the hydroxylation of proline (Pro residues in their triple-helical domain repeating sequence Xaa-Pro-Gly to function properly as a main structural component of the extracellular matrix in animals at physiologically relevant conditions. The regioselective proline hydroxylation is catalyzed by a specific prolyl 4-hydroxylase (P4H as a posttranslational processing step. Results A recombinant human collagen type I α-1 (rCIα1 with high percentage of hydroxylated prolines (Hyp was produced in transgenic maize seeds when co-expressed with both the α- and β- subunits of a recombinant human P4H (rP4H. Germ-specific expression of rCIα1 using maize globulin-1 gene promoter resulted in an average yield of 12 mg/kg seed for the full-length rCIα1 in seeds without co-expression of rP4H and 4 mg/kg seed for the rCIα1 (rCIα1-OH in seeds with co-expression of rP4H. High-resolution mass spectrometry (HRMS analysis revealed that nearly half of the collagenous repeating triplets in rCIα1 isolated from rP4H co-expressing maize line had the Pro residues changed to Hyp residues. The HRMS analysis determined the Hyp content of maize-derived rCIα1-OH as 18.11%, which is comparable to the Hyp level of yeast-derived rCIα1-OH (17.47% and the native human CIa1 (14.59%, respectively. The increased Hyp percentage was correlated with a markedly enhanced thermal stability of maize-derived rCIα1-OH when compared to the non-hydroxylated rCIα1. Conclusions This work shows that maize has potential to produce adequately modified exogenous proteins with mammalian-like post-translational modifications that may be require for their use as pharmaceutical and industrial products.

  18. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yu, E-mail: xuyu1001@gmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Liu, Zhengchun, E-mail: l135027@126.com [Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Kong, Haiyan, E-mail: suppleant@163.com [Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Sun, Wenjie, E-mail: wendy11240325@163.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Liao, Zhengkai, E-mail: fastbeta@gmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Zhou, Fuxiang, E-mail: happyzhoufx@sina.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Xie, Conghua, E-mail: chxie_65@hotmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); and others

    2011-09-09

    Highlights: {yields} A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. {yields} The promoter was characterized with radiation-inducibility and tumor-specificity. {yields} Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. {yields} Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  19. Gene co-expression analysis identifies brain regions and cell types involved in migraine pathophysiology: a GWAS-based study using the Allen Human Brain Atlas.

    Science.gov (United States)

    Eising, Else; Huisman, Sjoerd M H; Mahfouz, Ahmed; Vijfhuizen, Lisanne S; Anttila, Verneri; Winsvold, Bendik S; Kurth, Tobias; Ikram, M Arfan; Freilinger, Tobias; Kaprio, Jaakko; Boomsma, Dorret I; van Duijn, Cornelia M; Järvelin, Marjo-Riitta R; Zwart, John-Anker; Quaye, Lydia; Strachan, David P; Kubisch, Christian; Dichgans, Martin; Davey Smith, George; Stefansson, Kari; Palotie, Aarno; Chasman, Daniel I; Ferrari, Michel D; Terwindt, Gisela M; de Vries, Boukje; Nyholt, Dale R; Lelieveldt, Boudewijn P F; van den Maagdenberg, Arn M J M; Reinders, Marcel J T

    2016-04-01

    Migraine is a common disabling neurovascular brain disorder typically characterised by attacks of severe headache and associated with autonomic and neurological symptoms. Migraine is caused by an interplay of genetic and environmental factors. Genome-wide association studies (GWAS) have identified over a dozen genetic loci associated with migraine. Here, we integrated migraine GWAS data with high-resolution spatial gene expression data of normal adult brains from the Allen Human Brain Atlas to identify specific brain regions and molecular pathways that are possibly involved in migraine pathophysiology. To this end, we used two complementary methods. In GWAS data from 23,285 migraine cases and 95,425 controls, we first studied modules of co-expressed genes that were calculated based on human brain expression data for enrichment of genes that showed association with migraine. Enrichment of a migraine GWAS signal was found for five modules that suggest involvement in migraine pathophysiology of: (i) neurotransmission, protein catabolism and mitochondria in the cortex; (ii) transcription regulation in the cortex and cerebellum; and (iii) oligodendrocytes and mitochondria in subcortical areas. Second, we used the high-confidence genes from the migraine GWAS as a basis to construct local migraine-related co-expression gene networks. Signatures of all brain regions and pathways that were prominent in the first method also surfaced in the second method, thus providing support that these brain regions and pathways are indeed involved in migraine pathophysiology.

  20. Co-Expression of Monodehydroascorbate Reductase and Dehydroascorbate Reductase from Brassica rapa Effectively Confers Tolerance to Freezing-Induced Oxidative Stress

    Science.gov (United States)

    Shin, Sun-Young; Kim, Myung-Hee; Kim, Yul-Ho; Park, Hyang-Mi; Yoon, Ho-Sung

    2013-01-01

    Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing. PMID:24170089

  1. Mini-P-gp and P-gp Co-Expression in Brown Trout Erythrocytes: A Prospective Blood Biomarker of Aquatic Pollution

    Science.gov (United States)

    Valton, Emeline; Amblard, Christian; Desmolles, François; Combourieu, Bruno; Penault-Llorca, Frédérique; Bamdad, Mahchid

    2015-01-01

    In aquatic organisms, such as fish, blood is continually exposed to aquatic contaminants. Multidrug Resistance (MDR) proteins are ubiquitous detoxification membrane pumps, which recognize various xenobiotics. Moreover, their expression is induced by a large class of drugs and pollutants. We have highlighted the co-expression of a mini P-gp of 75 kDa and a P-gp of 140 kDa in the primary culture of brown trout erythrocytes and in the erythrocytes of wild brown trout collected from three rivers in the Auvergne region of France. In vitro experiments showed that benzo[a]pyrene, a highly toxic pollutant model, induced the co-expression of mini-P-gp and P-gp in trout erythrocytes in a dose-dependent manner and relay type response. Similarly, in the erythrocytes of wild brown trout collected from rivers contaminated by a mixture of PAH and other multi-residues of pesticides, mini-P-gp and P-gp were able to modulate their expression, according to the nature of the pollutants. The differential and complementary responses of mini-P-gp and P-gp in trout erythrocytes suggest the existence in blood cells of a real protective network against xenobiotics/drugs. This property could be exploited to develop a blood biomarker of river pollution. PMID:26854141

  2. Neuronal ceroid lipofuscinosis genes, CLN2, CLN3 and CLN5 are spatially and temporally co-expressed in a developing mouse brain.

    Science.gov (United States)

    Fabritius, A-L; Vesa, J; Minye, H M; Nakano, I; Kornblum, H; Peltonen, L

    2014-12-01

    Neuronal ceroid lipofuscinosis (NCL) diseases consist of a group of genetically inherited neurodegenerative disorders that share common symptoms such as seizures, psychomotor retardation, blindness, and premature death. Although gene defects behind the NCL diseases are well characterized, very little is known how these defects affect normal development of the brain and cause the pathology of the disease. To obtain understanding of the development of the cell types that are mostly affected by defective function of CLN proteins, timing of expression of CLN2, CLN3 and CLN5 genes was investigated in developing mouse brain. The relationship between the expression pattern and the developmental stage of the brain showed that these genes are co-expressed spatially and temporally during brain development. Throughout the development strong expression of the three mRNAs was detected in germinal epithelium and in ventricle regions, hippocampus and cerebellum, all representing regions that are known to be associated with the formation of new neurons. More specifically, RT-PCR studies on developing mouse cortices revealed that the CLN genes were temporally co-expressed in the neural progenitor cells together with known stem cell markers. This suggested that CLN2, CLN3 and CLN5 genes may play an important role in early embryonal neurogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Co-expression of HIV-1 virus-like particles and granulocyte-macrophage colony stimulating factor by GEO-D03 DNA vaccine.

    Science.gov (United States)

    Hellerstein, Michael; Xu, Yongxian; Marino, Tracie; Lu, Shan; Yi, Hong; Wright, Elizabeth R; Robinson, Harriet L

    2012-11-01

    Here, we report on GEO-D03, a DNA vaccine that co-expresses non-infectious HIV-1 virus-like particles (VLPs) and the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). The virus-like particles display the native gp160 form of the HIV-1 Envelope glycoprotein (Env) and are designed to elicit antibody against the natural form of Env on virus and virus-infected cells. The DNA-expressed HIV Gag, Pol and Env proteins also have the potential to elicit virus-specific CD4 and CD8 T cells. The purpose of the co-expressed GM-CSF is to target a cytokine that recruits, expands and differentiates macrophages and dendritic cells to the site of VLP expression. The GEO-D03 DNA vaccine is currently entered into human trials as a prime for a recombinant modified vaccinia Ankara (MVA) boost. In preclinical studies in macaques using an SIV prototype vaccine, this vaccination regimen elicited both anti-viral T cells and antibody, and provided 70% protection against acquisition during 12 weekly rectal exposures with a heterologous SIV. Higher avidity of the Env-specific Ab for the native form of the Env in the challenge virus correlated with lower likelihood of SIV infection.

  4. Comprehensive meta-analysis, co-expression, and miRNA nested network analysis identifies gene candidates in citrus against Huanglongbing disease.

    Science.gov (United States)

    Rawat, Nidhi; Kiran, Sandhya P; Du, Dongliang; Gmitter, Fred G; Deng, Zhanao

    2015-07-28

    Huanglongbing (HLB), the most devastating disease of citrus, is associated with infection by Candidatus Liberibacter asiaticus (CaLas) and is vectored by the Asian citrus psyllid (ACP). Recently, the molecular basis of citrus-HLB interactions has been examined using transcriptome analyses, and these analyses have identified many probe sets and pathways modulated by CaLas infection among different citrus cultivars. However, lack of consistency among reported findings indicates that an integrative approach is needed. This study was designed to identify the candidate probe sets in citrus-HLB interactions using meta-analysis and gene co-expression network modelling. Twenty-two publically available transcriptome studies on citrus-HLB interactions, comprising 18 susceptible (S) datasets and four resistant (R) datasets, were investigated using Limma and RankProd methods of meta-analysis. A combined list of 7,412 differentially expressed probe sets was generated using a Teradata in-house Structured Query Language (SQL) script. We identified the 65 most common probe sets modulated in HLB disease among different tissues from the S and R datasets. Gene ontology analysis of these probe sets suggested that carbohydrate metabolism, nutrient transport, and biotic stress were the core pathways that were modulated in citrus by CaLas infection and HLB development. We also identified R-specific probe sets, which encoded leucine-rich repeat proteins, chitinase, constitutive disease resistance (CDR), miraculins, and lectins. Weighted gene co-expression network analysis (WGCNA) was conducted on 3,499 probe sets, and 21 modules with major hub probe sets were identified. Further, a miRNA nested network was created to examine gene regulation of the 3,499 target probe sets. Results suggest that csi-miR167 and csi-miR396 could affect ion transporters and defence response pathways, respectively. Most of the potential candidate hub probe sets were co-expressed with gibberellin pathway (GA

  5. Multi-tissue analysis of co-expression networks by higher-order generalized singular value decomposition identifies functionally coherent transcriptional modules.

    Science.gov (United States)

    Xiao, Xiaolin; Moreno-Moral, Aida; Rotival, Maxime; Bottolo, Leonardo; Petretto, Enrico

    2014-01-01

    Recent high-throughput efforts such as ENCODE have generated a large body of genome-scale transcriptional data in multiple conditions (e.g., cell-types and disease states). Leveraging these data is especially important for network-based approaches to human disease, for instance to identify coherent transcriptional modules (subnetworks) that can inform functional disease mechanisms and pathological pathways. Yet, genome-scale network analysis across conditions is significantly hampered by the paucity of robust and computationally-efficient methods. Building on the Higher-Order Generalized Singular Value Decomposition, we introduce a new algorithmic approach for efficient, parameter-free and reproducible identification of network-modules simultaneously across multiple conditions. Our method can accommodate weighted (and unweighted) networks of any size and can similarly use co-expression or raw gene expression input data, without hinging upon the definition and stability of the correlation used to assess gene co-expression. In simulation studies, we demonstrated distinctive advantages of our method over existing methods, which was able to recover accurately both common and condition-specific network-modules without entailing ad-hoc input parameters as required by other approaches. We applied our method to genome-scale and multi-tissue transcriptomic datasets from rats (microarray-based) and humans (mRNA-sequencing-based) and identified several common and tissue-specific subnetworks with functional significance, which were not detected by other methods. In humans we recapitulated the crosstalk between cell-cycle progression and cell-extracellular matrix interactions processes in ventricular zones during neocortex expansion and further, we uncovered pathways related to development of later cognitive functions in the cortical plate of the developing brain which were previously unappreciated. Analyses of seven rat tissues identified a multi-tissue subnetwork of co-expressed

  6. Multi-tissue analysis of co-expression networks by higher-order generalized singular value decomposition identifies functionally coherent transcriptional modules.

    Directory of Open Access Journals (Sweden)

    Xiaolin Xiao

    2014-01-01

    Full Text Available Recent high-throughput efforts such as ENCODE have generated a large body of genome-scale transcriptional data in multiple conditions (e.g., cell-types and disease states. Leveraging these data is especially important for network-based approaches to human disease, for instance to identify coherent transcriptional modules (subnetworks that can inform functional disease mechanisms and pathological pathways. Yet, genome-scale network analysis across conditions is significantly hampered by the paucity of robust and computationally-efficient methods. Building on the Higher-Order Generalized Singular Value Decomposition, we introduce a new algorithmic approach for efficient, parameter-free and reproducible identification of network-modules simultaneously across multiple conditions. Our method can accommodate weighted (and unweighted networks of any size and can similarly use co-expression or raw gene expression input data, without hinging upon the definition and stability of the correlation used to assess gene co-expression. In simulation studies, we demonstrated distinctive advantages of our method over existing methods, which was able to recover accurately both common and condition-specific network-modules without entailing ad-hoc input parameters as required by other approaches. We applied our method to genome-scale and multi-tissue transcriptomic datasets from rats (microarray-based and humans (mRNA-sequencing-based and identified several common and tissue-specific subnetworks with functional significance, which were not detected by other methods. In humans we recapitulated the crosstalk between cell-cycle progression and cell-extracellular matrix interactions processes in ventricular zones during neocortex expansion and further, we uncovered pathways related to development of later cognitive functions in the cortical plate of the developing brain which were previously unappreciated. Analyses of seven rat tissues identified a multi

  7. [Construction and identification of non-replication recombinant vaccinia virus co-expressing human papillomavirus type 16 L1/L2/E6/E7 proteins].

    Science.gov (United States)

    Huang, Wei; Tian, Hou-wen; Ren, Jiao; Fan, Jiang-tao; Zhao, Li; Bian, Tao; Lu, Zhen-hua; Ruan, Li

    2005-09-01

    To generate a human papillomavirus (HPV16) prophylactic and therapeutic vaccine candidate for cervical cancer. HPV16 major capsid protein L1 gene/minor capsid protein L2 gene and HPV16 early E6/E7 genes were inserted into a vaccinia virus expression vector. A strain of non-recombinant vaccinia virus containing the sequences was obtained through a homologous recombination and identified. DNA hybridization confirmed that the HPV16L1/L2/E6/E7 genes were integrated into vaccinia virus DNA. Western Blot result showed that full-length L1/L2/E6/E7 proteins were co-expressed in CEF cells infected with the recombinant virus. NTVJE6E7CKL1L2 could be taken as a candidate of prophylactic and therapeutic vaccine for HPV-associated tumors and their precancerous transformations.

  8. Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP fused antigens: a potential tool to develop DNA vaccines against flaviviruses

    Directory of Open Access Journals (Sweden)

    Rafael Dhalia

    2009-12-01

    Full Text Available Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the developent of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP. The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.A vacinação é a estratégia mais prática e o melhor custo-benefício para prevenir a maioria das infecções dos flavivirus, para os quais existe vacina disponível. Entretanto, as vacinas baseadas em vírus atenuados podem potencialmente promover efeitos colaterais e, mais raramente, reações fatais. Diante deste cenário, o desenvolvimento de estratégias alternativas de vacinação, como vacinas baseadas em DNA codificando seqüências específicas dos flavivirus, está sendo considerado

  9. Co-expression of Bacillus thuringiensis Cry4Ba and Cyt2Aa2 in Escherichia coli revealed high synergism against Aedes aegypti and Culex quinquefasciatus larvae.

    Science.gov (United States)

    Promdonkoy, Boonhiang; Promdonkoy, Patcharee; Panyim, Sakol

    2005-11-01

    Cry4Ba is a delta-endotoxin produced by Bacillus thuringiensis subsp. israelensis and Cyt2Aa2 is a cytolytic delta-endotoxin produced by B. thuringiensis subsp. darmstadiensis. Cry4Ba produced in Escherichia coli was toxic to Aedes aegypti larvae (LC(50)=140 ng ml(-1)) but virtually inactive to Culex quinquefasciatus larvae. Cyt2Aa2 expressed in E. coli exhibited moderate activity against A. aegypti and C. quinquefasciatus larvae with LC(50) values of 350 and 250 ng ml(-1), respectively. Co-expression of both toxins in E. coli dramatically increased toxicity to both A. aegypti andC. quinquefasciatus larvae (LC(50)=7 and 20 ng ml(-1), respectively). This is the first report to demonstrate that Cry4Ba and Cyt2Aa2 have high synergistic activity against C. quinquefasciatus larvae.

  10. Weighted gene co-expression network analysis of expression data of monozygotic twins identifies specific modules and hub genes related to BMI

    DEFF Research Database (Denmark)

    Wang, Weijing; Jiang, Wenjie; Hou, Lin

    2017-01-01

    .04) and disease status (r = 0.56, P = 0.04). Categories of positive regulation of phospholipase activity, high-density lipoprotein particle clearance, chylomicron remnant clearance, reverse cholesterol transport, intermediate-density lipoprotein particle, chylomicron, low-density lipoprotein particle, very...... and weighted gene co-expression network analysis (WGCNA) to identify significant genes and specific modules related to BMI based on gene expression profile data of 7 discordant monozygotic twins. RESULTS: In the differential gene expression analysis, it appeared that 32 differentially expressed genes (DEGs......-low-density lipoprotein particle, voltage-gated potassium channel complex, cholesterol transporter activity, and neuropeptide hormone activity were significantly enriched within GO database for this module. And alcoholism and cell adhesion molecules pathways were significantly enriched within KEGG database. Several hub...

  11. Co-expression of an alcohol dehydrogenase and a cyclohexanone monooxygenase for cascade reactions facilitates the regeneration of the NADPH cofactor.

    Science.gov (United States)

    Kohl, Anna; Srinivasamurthy, Vishnu; Böttcher, Dominique; Kabisch, Johannes; Bornscheuer, Uwe T

    2018-01-01

    The introduction of a three-enzyme cascade (comprising a cyclohexanone monooxygenase (CHMO), an alcohol dehydrogenase (ADH) and a lipase (CAL-A)) for the production of oligo-ε-caprolactone provided self-sufficiency with respect to NADPH-cofactor regeneration and reduced inhibiting effects on the central CHMO enzyme. For further optimization of cofactor regeneration, now a co-expression of CHMO and ADH in E. coli using a Duet™ vector was performed. This led to higher conversion values of the substrate cyclohexanol in whole-cell biocatalysis compared to an expression of both enzymes from two separate plasmids. Furthermore, a more advantageous balance of expression levels between the partial cascade enzymes was achieved via engineering of the ribosome binding site. This contributed to an even faster cofactor regeneration rate. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Transient co-expression for fast and high-yield production of antibodies with human-like N-glycans in plants.

    Science.gov (United States)

    Vézina, Louis-P; Faye, Loïc; Lerouge, Patrice; D'Aoust, Marc-André; Marquet-Blouin, Estelle; Burel, Carole; Lavoie, Pierre-Olivier; Bardor, Muriel; Gomord, Véronique

    2009-06-01

    Plant-based transient expression is potentially the most rapid and cost-efficient system for the production of recombinant pharmaceutical proteins, but safety concerns associated with plant-specific N-glycosylation have hampered its adoption as a commercial production system. In this article, we describe an approach based on the simultaneous transient co-expression of an antibody, a suppressor of silencing and a chimaeric human beta1,4-galactosyltransferase targeted for optimal activity to the early secretory pathway in agroinfiltrated Nicotiana benthamiana leaves. This strategy allows fast and high-yield production of antibodies with human-like N-glycans and, more generally, provides solutions to many critical problems posed by the large-scale production of therapeutic and vaccinal proteins, specifically yield, volume and quality.

  13. Whole-cell bioreduction of aromatic α-keto esters using Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase co-expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Egger Sigrid

    2008-12-01

    Full Text Available Abstract Background Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful reductases are expressed heterologously in Escherichia coli to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in E. coli usually requires that reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP+ or NAD+ dependent dehydrogenase that is co-expressed. Candida tenuis xylose reductase (CtXR was previously shown to promote NADH dependent reduction of aromatic α-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an E. coli strain co-expressing CtXR and formate dehydrogenase from Candida boidinii (CbFDH. The bacterial system was evaluated for the synthesis of ethyl R-4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from Saccharomyces cerevisiae expressing CtXR. Results Gene co-expression from a pETDuet-1 vector yielded about 260 and 90 units of intracellular CtXR and CbFDH activity per gram of dry E. coli cell mass (gCDW. The maximum conversion rate (rS for ethyl 4-cyanobenzoylformate by intact or polymyxin B sulphate-permeabilized cells was similar (2 mmol/gCDWh, suggesting that the activity of CbFDH was partly rate-limiting overall. Uncatalyzed ester hydrolysis in substrate as well as inactivation of CtXR and CbFDH in the presence of the α-keto ester constituted major restrictions to the yield of alcohol product. Using optimized reaction conditions (100 mM substrate; 40 gCDW/L, we obtained ethyl R-4-cyanomandelate with an enantiomeric excess (e.e. of 97.2% in a yield of 82

  14. In silico identification of miRNAs and their target genes and analysis of gene co-expression network in saffron (Crocus sativus L.) stigma.

    Science.gov (United States)

    Zinati, Zahra; Shamloo-Dashtpagerdi, Roohollah; Behpouri, Ali

    2016-12-01

    As an aromatic and colorful plant of substantive taste, saffron (Crocus sativus L.) owes such properties of matter to growing class of the secondary metabolites derived from the carotenoids, apocarotenoids. Regarding the critical role of microRNAs in secondary metabolic synthesis and the limited number of identified miRNAs in C. sativus, on the other hand, one may see the point how the characterization of miRNAs along with the corresponding target genes in C. sativus might expand our perspectives on the roles of miRNAs in carotenoid/apocarotenoid biosynthetic pathway. A computational analysis was used to identify miRNAs and their targets using EST (Expressed Sequence Tag) library from mature saffron stigmas. Then, a gene co- expression network was constructed to identify genes which are potentially involved in carotenoid/apocarotenoid biosynthetic pathways. EST analysis led to the identification of two putative miRNAs (miR414 and miR837-5p) along with the corresponding stem- looped precursors. To our knowledge, this is the first report on miR414 and miR837-5p in C. sativus. Co-expression network analysis indicated that miR414 and miR837-5p may play roles in C. sativus metabolic pathways and led to identification of candidate genes including six transcription factors and one protein kinase probably involved in carotenoid/apocarotenoid biosynthetic pathway. Presence of transcription factors, miRNAs and protein kinase in the network indicated multiple layers of regulation in saffron stigma. The candidate genes from this study may help unraveling regulatory networks underlying the carotenoid/apocarotenoid biosynthesis in saffron and designing metabolic engineering for enhanced secondary metabolites.

  15. Co-expression of alpha-1 antitrypsin with cytoplasmic domain of v-SNARE in Pichia pastoris: preserving biological activity of alpha-1 antitrypsin.

    Science.gov (United States)

    Khatami, Maryam; Hosseini, Seyed Nezamedin; Hasannia, Sadegh

    2017-08-01

    Alpha-1-antitrypsin (A1AT) is a major serum protein in human with protease inhibitory activity. Because of its extensive application in medicine, recombinant DNA technology has been considered for its production. The current study is going to examine co-expression of A1AT, and soluble domain of v-SNARE in Pichia pastoris, which can prevent the secretion of A1AT after passing the secretory pathway thoroughly. This was done mainly to preserve the biological activity of A1AT which in the secretory mode might be impaired in the fermentation and early clarification conditions. SNARE proteins are the driving force for vesicle docking and membrane fusion in the term defined as exocytosis. Intracellular expression of the cytoplasmic domain of v-SNARE and its subsequent interaction to form SNARE complex can intensify the competition for A1AT secretory vesicles to be fused and released to the media. Our investigation shows successful co-expression of A1AT in the form of post-Golgi vesicles (PGVs), and the cytoplasmic domain of v-SNARE. Our findings confirmed the reduction of A1AT secretion by 45% which trapped in post-Golgi A1AT secretory vesicles inside the cell were biologically more active than secreted A1AT protein. These results indicate that the inhibition of A1AT secretion can protect its biological activity in fermentation and clarification processes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. The variant hERG/R148W associated with LQTS is a mutation that reduces current density on co-expression with the WT.

    Science.gov (United States)

    Mechakra, Asma; Vincent, Yohann; Chevalier, Philippe; Millat, Gilles; Ficker, Eckhard; Jastrzebski, Marek; Poulin, Hugo; Pouliot, Valérie; Chahine, Mohamed; Christé, Georges

    2014-02-25

    A variant of the ether-à-go-go related channel (hERG), p.Arg148Trp (R148W) was found at heterozygous state in two infants who died from sudden infant death syndrome (SIDS), one with documented prolonged QTc and Torsade de Pointes (TdP), and in an adult woman with QTc >500 ms, atrioventricular block and TdP. This variant was previously reported in cases of severe ventricular arrhythmia but very rarely in control subjects. Its classification as mutation or polymorphism awaited electrophysiological characterization. The properties of this N-terminal, proximal domain, hERG variant were explored in Xenopus oocytes injected with the same amount of RNA encoding for either hERG/WT or hERG/R148W or their equimolar mixture. The human ventricular cell (TNNP) model was used to test the effects of changes in hERG current. R148W alone produced a current similar to the WT (369 ± 76 nA (mean ± SEM), n=13 versus 342 ± 55 nA in WT, n=13), while the co-expression of 1/2 WT+1/2 R148W lowered the current by 29% versus WT (243 ± 35 nA, n=13, phERG current as evidenced here when co-expressing the hERG/R148W variant with the WT may have predisposed to the observed long QT syndrome and associated TdP. Therefore, the heterozygous carriers of hERG/R148W may be at risk of cardiac sudden death. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Co-expression of march5b and tlr7 in large yellow croaker Larimichthys crocea in response to Cryptocaryon irritans infection.

    Science.gov (United States)

    Zhang, D L; Yu, D H; Chen, J; Chen, C; Wang, Z Y

    2015-08-01

    In this study, molecular characteristics of march5b and co-expression of march5b and tlr7 in response to the infection of Cryptocaryon irritans in the large yellow croaker Larimichthys crocea were investigated. The full-length complementary (c)DNA of march5b was 1314 bp, including an open reading frame of 846 bp encoding a polypeptide of 281 amino acids, and the full-length genomic sequence was composed of 23,577 nucleotides, including six exons and five introns. The putative March5b protein contained a RINGv motif and four transmembrane domains. The march5b transcripts were broadly distributed in all detected tissues, with a strong expression in blood, brain and gills, and a weak expression in kidney by quantitative PCR analysis. The expression of march5b and tlr7 in the skin, gills, spleen and head kidney changed in the same manner at most time points post-primary infection with C. irritans. Significant increase was observed in the skin with march5b at days 2 and 3 by 26.10 and 6.88 fold, respectively, and with tlr7 at day 3 by 57.68 fold, when compared with the control. Their expressions, however, were decreased in the gills, especially at day 3 (march5b by 8.9%, tlr7 by 22.06%). In the spleen and head kidney, march5b and tlr7 transcripts were up-regulated early, then noticeably declined at day 3. These results suggested that march5b and tlr7 are co-expressed in response to parasite infection and March5b probably catalyses ubiquitination of some proteins of TLR7 signalling pathway. © 2015 The Fisheries Society of the British Isles.

  18. Co-expression of vascular and lymphatic endothelial cell markers on early endothelial cells present in aspirated coronary thrombi from patients with ST-elevation myocardial infarction.

    Science.gov (United States)

    Rakocevic, Jelena; Kojic, Snezana; Orlic, Dejan; Stankovic, Goran; Ostojic, Miodrag; Petrovic, Olga; Zaletel, Ivan; Puskas, Nela; Todorovic, Vera; Labudovic-Borovic, Milica

    2016-02-01

    Angiogenesis is the growth of both new vascular and lymphatic blood vessels from the existing vasculature. During this process, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs) express specific markers, which help their discrimination and easier identification. Since the coronary thrombi material aspirated from patients with ST-elevation myocardial infarction (STEMI) proved as good angiogenesis model, we investigated the expression of CD34 and CD31 as BECs markers, and D2-40, LYVE-1 and VEGFR3 as LEC markers in this material. Aspirated thrombi were stained immunohistochemically for CD34, CD31, D2-40, LYVE-1 and VEGFR3. Organizational patterns of immunopositive cells were graded as single cells, clusters or microvessels. Double immunofluorescence for CD31, D2-40, LYVE-1 and VEGRF3 was done. Thrombi were also graded as fresh (5days old). Serial sections of aspirated thrombi showed concordant BEC and LEC markers immunopositivity. Double immunoflorescence proved co-expression of CD31 and LEC markers on the same cells. Cells expressing LEC markers organized in clusters and microvessels were mainly present in lytic and organized thrombi. Co-expression of BEC and LEC markers on the same non-tumorous cell during thrombus neovascularization indicates existing in vivo plasticity of endothelial cells under non-tumorous pathological conditions. It also points that CD34 and CD31 on one hand, and D2-40, LYVE-1 and VEGFR3 immunostaining on the other hand, cannot solely be a reliable indicators whether vessel is lymphatic or not. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Cytotoxic effect of co-expression of human hepatitis A virus 3C protease and bifunctional suicide protein FCU1 genes in a bicistronic vector.

    Science.gov (United States)

    Komissarov, Alexey; Demidyuk, Ilya; Safina, Dina; Roschina, Marina; Shubin, Andrey; Lunina, Nataliya; Karaseva, Maria; Kostrov, Sergey

    2017-08-01

    Recent reports on various cancer models demonstrate a great potential of cytosine deaminase/5-fluorocytosine suicide system in cancer therapy. However, this approach has limited success and its application to patients has not reached the desirable clinical significance. Accordingly, the improvement of this suicide system is an actively developing trend in gene therapy. The purpose of this study was to explore the cytotoxic effect observed after co-expression of hepatitis A virus 3C protease (3C) and yeast cytosine deaminase/uracil phosphoribosyltransferase fusion protein (FCU1) in a bicistronic vector. A set of mono- and bicistronic plasmid constructs was generated to provide individual or combined expression of 3C and FCU1. The constructs were introduced into HEK293 and HeLa cells, and target protein synthesis as well as the effect of 5-fluorocytosine on cell death and the time course of the cytotoxic effect was studied. The obtained vectors provide for the synthesis of target proteins in human cells. The expression of the genes in a bicistronic construct provide for the cytotoxic effect comparable to that observed after the expression of genes in monocistronic constructs. At the same time, co-expression of FCU1 and 3C recapitulated their cytotoxic effects. The combined effect of the killer and suicide genes was studied for the first time on human cells in vitro. The integration of different gene therapy systems inducing cell death (FCU1 and 3C genes) in a bicistronic construct allowed us to demonstrate that it does not interfere with the cytotoxic effect of each of them. A combination of cytotoxic genes in multicistronic vectors can be used to develop pluripotent gene therapy agents.

  20. Co-expression of Exo-inulinase and Endo-inulinase Genes in the Oleaginous Yeast Yarrowia lipolytica for Efficient Single Cell Oil Production from Inulin.

    Science.gov (United States)

    Shi, Nianci; Mao, Weian; He, Xiaoxia; Chi, Zhe; Chi, Zhenming; Liu, Guanglei

    2017-11-18

    Yarrowia lipolytica is a promising platform for the single cell oil (SCO) production. In this study, a transformant X+N8 in which exo- and endo-inulinase genes were co-expressed could produce an inulinase activity of 124.33 U/mL within 72 h. However, the inulinase activity of a transformant X2 carrying a single exo-inulinase gene was only 47.33 U/mL within 72 h. Moreover, the transformant X+N8 could accumulate 48.13% (w/w) SCO from inulin and the cell dry weight reached 13.63 g/L within 78 h, which were significantly higher than those of the transformant X2 (41.87% (w/w) and 11.23 g/L) under the same conditions. In addition, inulin hydrolysis and utilization of the transformant X+N8 were also more efficient than those of the transformant X2 during the fermentation process. These results demonstrated that the co-expression of the exo- and endo-inulinase genes significantly enhanced the SCO production from inulin due to the improvement of the inulinase activity and the synergistic action of exo- and endo-inulinase. Besides, over 95.01% of the fatty acids from the transformant X+N8 were C16-C18, especially C18:1 (53.10%), suggesting that the fatty acids could be used as feedstock for biodiesel production.

  1. Microarray profiling and co-expression network analysis of circulating lncRNAs and mRNAs associated with major depressive disorder.

    Directory of Open Access Journals (Sweden)

    Zhifen Liu

    Full Text Available LncRNAs, which represent one of the most highly expressed classes of ncRNAs in the brain, are becoming increasingly interesting with regard to brain functions and disorders. However, changes in the expression of regulatory lncRNAs in Major Depressive Disorder (MDD have not yet been reported. Using microarrays, we profiled the expression of 34834 lncRNAs and 39224 mRNAs in peripheral blood sampled from MDD patients as well as demographically-matched controls. Among these, we found that 2007 lncRNAs and 1667 mRNAs were differentially expressed, 17 of which were documented as depression-related gene in previous studies. Gene Ontology (GO and pathway analyses indicated that the biological functions of differentially expressed mRNAs were related to fundamental metabolic processes and neurodevelopment diseases. To investigate the potential regulatory roles of the differentially expressed lncRNAs on the mRNAs, we also constructed co-expression networks composed of the lncRNAs and mRNAs, which shows significant correlated patterns of expression. In the MDD-derived network, there were a greater number of nodes and connections than that in the control-derived network. The lncRNAs located at chr10:874695-874794, chr10:75873456-75873642, and chr3:47048304-47048512 may be important factors regulating the expression of mRNAs as they have previously been reported associations with MDD. This study is the first to explore genome-wide lncRNA expression and co-expression with mRNA patterns in MDD using microarray technology. We identified circulating lncRNAs that are aberrantly expressed in MDD and the results suggest that lncRNAs may contribute to the molecular pathogenesis of MDD.

  2. Efficient androst-1,4-diene-3,17-dione production by co-expressing 3-ketosteroid-Δ1 -dehydrogenase and catalase in Bacillus subtilis.

    Science.gov (United States)

    Shao, M; Sha, Z; Zhang, X; Rao, Z; Xu, M; Yang, T; Xu, Z; Yang, S

    2017-01-01

    3-ketosteroid-Δ1 -dehydrogenase (KSDD), a flavin adenine dinucleotide (FAD)-dependent enzyme involved in sterol metabolism, specifically catalyses the conversion of androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). However, the low KSDD activity and the toxic effects of hydrogen peroxide (H2 O2 ) generated during the biotransformation of AD to ADD with FAD regeneration hinder its application on AD conversion. The aim of this work was to improve KSDD activity and eliminate the toxic effects of the generated H2 O2 to enhance ADD production. The ksdd gene obtained from Mycobacterium neoaurum JC-12 was codon-optimized to increase its expression level in Bacillus subtilis, and the KSDD activity reached 12·3 U mg-1 , which was sevenfold of that of codon-unoptimized gene. To improve AD conversion, catalase was co-expressed with KSDD in B. subtilis 168/pMA5-ksddopt -katA to eliminate the toxic effects of H2 O2 generated during AD conversion. Finally, under optimized bioconversion conditions, fed-batch strategy was carried out and the ADD yield improved to 8·76 g l-1 . This work demonstrates the potential to improve enzyme activity by codon-optimization and eliminate the toxic effects of H2 O2 by co-expressing catalase. This study showed the highest ADD productivity ever reported and provides a promising strain for efficient ADD production in the pharmaceutical industry. © 2016 The Society for Applied Microbiology.

  3. GABAB1 and GABAB2 receptor subunits co-expressed in cultured human RPE cells regulate intracellular Ca2+ via Gi/o-protein and phospholipase C pathways.

    Science.gov (United States)

    Cheng, Z-Y; Wang, X-P; Schmid, K L; Han, X-G

    2014-11-07

    GABAB receptors associate with Gi/o-proteins that regulate voltage-gated Ca(2+) channels and thus the intracellular Ca(2+) concentration ([Ca(2+)]i), there is also reported cross-regulation of phospholipase C. These associations have been studied extensively in the brain and also shown to occur in non-neural cells (e.g. human airway smooth muscle). More recently GABAB receptors have been observed in chick retinal pigment epithelium (RPE). The aims were to investigate whether the GABAB receptor subunits, GABAB1 and GABAB2, are co-expressed in cultured human RPE cells, and then determine if the GABAB receptor similarly regulates the [Ca(2+)]i of RPE cells and if phospholipase C is involved. Human RPE cells were cultured from five donor eye cups. Evidence for GABAB1 and GABAB2 mRNAs and proteins in the RPE cell cultures was investigated using real time polymerase chain reaction, western blots and immunofluorescence. The effects of the GABAB receptor agonist baclofen, antagonist CGP46381, a Gi/o-protein inhibitor pertussis toxin, and the phospholipase C inhibitor U73122 on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo-3. Both GABAB1 and GABAB2 mRNA and protein were identified in cell cultures of human RPE; antibody staining was co-localized to the cell membrane and cytoplasm. One-hundred micromolars of baclofen caused a transient increase in the [Ca(2+)]i of RPE cells regardless of whether Ca(2+) was added to the buffer. Baclofen-induced increases in the [Ca(2+)]i were attenuated by pre-treatment with CGP46381, pertussis toxin, and U73122. GABAB1 and GABAB2 are co-expressed in cell cultures of human RPE. GABAB receptors in RPE regulate the [Ca(2+)]i via a Gi/o-protein and phospholipase C pathway. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  4. Integrating mRNA and miRNA Weighted Gene Co-Expression Networks with eQTLs in the Nucleus Accumbens of Subjects with Alcohol Dependence.

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    Mohammed Mamdani

    Full Text Available Alcohol consumption is known to lead to gene expression changes in the brain. After performing weighted gene co-expression network analyses (WGCNA on genome-wide mRNA and microRNA (miRNA expression in Nucleus Accumbens (NAc of subjects with alcohol dependence (AD; N = 18 and of matched controls (N = 18, six mRNA and three miRNA modules significantly correlated with AD were identified (Bonferoni-adj. p≤ 0.05. Cell-type-specific transcriptome analyses revealed two of the mRNA modules to be enriched for neuronal specific marker genes and downregulated in AD, whereas the remaining four mRNA modules were enriched for astrocyte and microglial specific marker genes and upregulated in AD. Gene set enrichment analysis demonstrated that neuronal specific modules were enriched for genes involved in oxidative phosphorylation, mitochondrial dysfunction and MAPK signaling. Glial-specific modules were predominantly enriched for genes involved in processes related to immune functions, i.e. cytokine signaling (all adj. p≤ 0.05. In mRNA and miRNA modules, 461 and 25 candidate hub genes were identified, respectively. In contrast to the expected biological functions of miRNAs, correlation analyses between mRNA and miRNA hub genes revealed a higher number of positive than negative correlations (χ2 test p≤ 0.0001. Integration of hub gene expression with genome-wide genotypic data resulted in 591 mRNA cis-eQTLs and 62 miRNA cis-eQTLs. mRNA cis-eQTLs were significantly enriched for AD diagnosis and AD symptom counts (adj. p = 0.014 and p = 0.024, respectively in AD GWAS signals in a large, independent genetic sample from the Collaborative Study on Genetics of Alcohol (COGA. In conclusion, our study identified putative gene network hubs coordinating mRNA and miRNA co-expression changes in the NAc of AD subjects, and our genetic (cis-eQTL analysis provides novel insights into the etiological mechanisms of AD.

  5. Weighted gene co-expression network analysis of expression data of monozygotic twins identifies specific modules and hub genes related to BMI.

    Science.gov (United States)

    Wang, Weijing; Jiang, Wenjie; Hou, Lin; Duan, Haiping; Wu, Yili; Xu, Chunsheng; Tan, Qihua; Li, Shuxia; Zhang, Dongfeng

    2017-11-13

    The therapeutic management of obesity is challenging, hence further elucidating the underlying mechanisms of obesity development and identifying new diagnostic biomarkers and therapeutic targets are urgent and necessary. Here, we performed differential gene expression analysis and weighted gene co-expression network analysis (WGCNA) to identify significant genes and specific modules related to BMI based on gene expression profile data of 7 discordant monozygotic twins. In the differential gene expression analysis, it appeared that 32 differentially expressed genes (DEGs) were with a trend of up-regulation in twins with higher BMI when compared to their siblings. Categories of positive regulation of nitric-oxide synthase biosynthetic process, positive regulation of NF-kappa B import into nucleus, and peroxidase activity were significantly enriched within GO database and NF-kappa B signaling pathway within KEGG database. DEGs of NAMPT, TLR9, PTGS2, HBD, and PCSK1N might be associated with obesity. In the WGCNA, among the total 20 distinct co-expression modules identified, coral1 module (68 genes) had the strongest positive correlation with BMI (r = 0.56, P = 0.04) and disease status (r = 0.56, P = 0.04). Categories of positive regulation of phospholipase activity, high-density lipoprotein particle clearance, chylomicron remnant clearance, reverse cholesterol transport, intermediate-density lipoprotein particle, chylomicron, low-density lipoprotein particle, very-low-density lipoprotein particle, voltage-gated potassium channel complex, cholesterol transporter activity, and neuropeptide hormone activity were significantly enriched within GO database for this module. And alcoholism and cell adhesion molecules pathways were significantly enriched within KEGG database. Several hub genes, such as GAL, ASB9, NPPB, TBX2, IL17C, APOE, ABCG4, and APOC2 were also identified. The module eigengene of saddlebrown module (212 genes) was also significantly

  6. ESR1 is co-expressed with closely adjacent uncharacterised genes spanning a breast cancer susceptibility locus at 6q25.1.

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    Anita K Dunbier

    2011-04-01

    Full Text Available Approximately 80% of human breast carcinomas present as oestrogen receptor α-positive (ER+ve disease, and ER status is a critical factor in treatment decision-making. Recently, single nucleotide polymorphisms (SNPs in the region immediately upstream of the ER gene (ESR1 on 6q25.1 have been associated with breast cancer risk. Our investigation of factors associated with the level of expression of ESR1 in ER+ve tumours has revealed unexpected associations between genes in this region and ESR1 expression that are important to consider in studies of the genetic causes of breast cancer risk. RNA from tumour biopsies taken from 104 postmenopausal women before and after 2 weeks treatment with an aromatase (oestrogen synthase inhibitor was analyzed on Illumina 48K microarrays. Multiple-testing corrected Spearman correlation revealed that three previously uncharacterized open reading frames (ORFs located immediately upstream of ESR1, C6ORF96, C6ORF97, and C6ORF211 were highly correlated with ESR1 (Rs =  0.67, 0.64, and 0.55 respectively, FDR<1 × 10(-7. Publicly available datasets confirmed this relationship in other groups of ER+ve tumours. DNA copy number changes did not account for the correlations. The correlations were maintained in cultured cells. An ERα antagonist did not affect the ORFs' expression or their correlation with ESR1, suggesting their transcriptional co-activation is not directly mediated by ERα. siRNA inhibition of C6ORF211 suppressed proliferation in MCF7 cells, and C6ORF211 positively correlated with a proliferation metagene in tumours. In contrast, C6ORF97 expression correlated negatively with the metagene and predicted for improved disease-free survival in a tamoxifen-treated published dataset, independently of ESR1. Our observations suggest that some of the biological effects previously attributed to ER could be mediated and/or modified by these co-expressed genes. The co-expression and function of these genes may be

  7. Whole number, distribution and co-expression of brn3 transcription factors in retinal ganglion cells of adult albino and pigmented rats.

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    Francisco M Nadal-Nicolás

    Full Text Available The three members of the Pou4f family of transcription factors: Pou4f1, Pou4f2, Pou4f3 (Brn3a, Brn3b and Brn3c, respectively play, during development, essential roles in the differentiation and survival of sensory neurons. The purpose of this work is to study the expression of the three Brn3 factors in the albino and pigmented adult rat. Animals were divided into these groups: i untouched; ii fluorogold (FG tracing from both superior colliculli; iii FG-tracing from one superior colliculus; iv intraorbital optic nerve transection or crush. All retinas were dissected as flat-mounts and subjected to single, double or triple immunohistofluorescence The total number of FG-traced, Brn3a, Brn3b, Brn3c or Brn3 expressing RGCs was automatically quantified and their spatial distribution assessed using specific routines. Brn3 factors were studied in the general RGC population, and in the intrinsically photosensitive (ip-RGCs and ipsilateral RGC sub-populations. Our results show that: i 70% of RGCs co- express two or three Brn3s and the remaining 30% express only Brn3a (26% or Brn3b; ii the most abundant Brn3 member is Brn3a followed by Brn3b and finally Brn3c; iii Brn3 a-, b- or c- expressing RGCs are similarly distributed in the retina; iv The vast majority of ip-RGCs do not express Brn3; v The main difference between both rat strains was found in the population of ipsilateral-RGCs, which accounts for 4.2% and 2.5% of the total RGC population in the pigmented and albino strain, respectively. However, more ipsilateral-RGCs express Brn3 factors in the albino than in the pigmented rat; vi RGCs that express only Brn3b and RGCs that co-express the three Brn3 members have the biggest nuclei; vii After axonal injury the level of Brn3a expression in the surviving RGCs decreases compared to control retinas. Finally, this work strengthens the validity of Brn3a as a marker to identify and quantify rat RGCs.

  8. Heterologous co-expression in E. coli of isoamylase genes from cassava Manihot esculenta Crantz 'KU50' achieves enzyme-active heteromeric complex formation.

    Science.gov (United States)

    Panpetch, Pawinee; Field, Robert A; Limpaseni, Tipaporn

    2018-01-29

    Cloning of two isoamylase genes, MeISA1 and MeISA2, from cassava (Manihot esculenta Crantz) tubers, accompanied by their co-expression in E. coli demonstrates a requirement for heteromeric complex formation to achieve debranching activity. Starch debranching enzyme (DBE) or isoamylase (ISA) (EC.3.2.1.68), an important enzyme in starch metabolism, catalyses the hydrolysis of α-1,6 glycosidic linkages of amylopectin. Isoforms of ISAs have been reported in higher plants and algae (Fujita et al. in Planta 208:283-293, 1999; Hussain et al. in Plant Cell 15:133-149, 2003; Ishizaki et al. in Agric Biol Chem 47:771-779, 1983; Mouille et al. in Plant Cell 8:1353-1366, 1996). In the current work, cassava ISA genes were isolated from cDNA generated from total RNA from tubers of Manihot esculanta Crantz cultivar KU50. MeISA1 and MeISA2 were successfully amplified and cloned into a pETDuet1 vector. The putative MeISA1 and MeISA2 proteins comprised 763 and 882 amino acids, with substantial similarity to StISA1 and StISA2 from potato (84.4% and 68.9%, respectively). Recombinant MeISA1 and MeISA2 were co-expressed in Escherichia coli SoluBL21 (DE3). HistrapTM-Purified rMeISA1 and rMeISA2 showed approximate molecular weights of 87 and 99 kDa, respectively, by SDS-PAGE. Debranching activity was only detectable in the column fractions where both recombinant ISA isoforms were present. The heteromeric DBE from crude extracts of 4-5 h induced cultures analysed by gel filtration chromatography and western blot showed combinations of rMeISA1 and rMeISA2 at ratios of 1:1 to 4:1. Pooled fractions with DBE activity were used for enzyme characterisation, which showed that the enzyme was specific for amylopectin, with optimum activity at 37 °C and pH 7.0. Enzyme activity was enhanced by Co2+, Mg2+ and Ca2+, but was strongly inhibited by Cu2+. Debranched amylopectin products showed chain length distributions typical of plant DBE.

  9. The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes

    KAUST Repository

    Meier, Stuart

    2010-01-26

    Background: Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3?,5?-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. Principal Findings: We have identified an Arabidopsis receptor type wall associated kinase-like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431-700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently coexpressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. Conclusions: We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP. © 2010 Meier et al.

  10. Co-expression of CD44 and ABCG2 in spheroid body-forming cells of gastric cancer cell line MKN45.

    Science.gov (United States)

    Liu, Jianming; Ma, Lilin; Xu, Junfei; Liu, Chun; Zhang, Jianguo; Liu, Jie; Chen, Ruixin; Zhou, Youlang

    2013-01-01

    The cancer stem cell (CSC) theory hypothesizes that CSCs are regarded as the cause of tumor formation, recurrence and metastasis. This study aimed to investigate whether spheroid body-forming cells in human gastric cancer cell were enriched for CSC properties, and to assess the expression of candidate CSC markers, cluster of differentiation 44 (CD44) and adenosine triphosphate binding cassette transporter G 2 (ABCG2) in the MKN45 spheroid body cells. Human gastric cancer cell line MKN45 were plated in stem cell conditioned culture system allowed for spheroid body forming. The expression levels of CD44 and ABCG2 in the spheroid body cells were assessed by quantitative real-time PCR, western blot analysis and immunofluorescence staining, and the tumorigenicity of the spheroid body-forming cells were assessed by in vivo xenograft studies in nude mice. The MKN45 cells could form spheroid bodies cultured in stem cell conditioned medium. The spheroid body-forming cells showed a significantly greater (p Spheroid body cells from gastric cancer cell line MKN45 cultured in stem cell conditioned medium possessed gastric CSC properties. The cells co-expressed of CD44 and ABCG2 might represent a subpopulation of gastric CSCs.

  11. A Network Approach of Gene Co-expression in the Zea mays/Aspergillus flavus Pathosystem to Map Host/Pathogen Interaction Pathways.

    Science.gov (United States)

    Musungu, Bryan M; Bhatnagar, Deepak; Brown, Robert L; Payne, Gary A; OBrian, Greg; Fakhoury, Ahmad M; Geisler, Matt

    2016-01-01

    A gene co-expression network (GEN) was generated using a dual RNA-seq study with the fungal pathogen Aspergillus flavus and its plant host Zea mays during the initial 3 days of infection. The analysis deciphered novel pathways and mapped genes of interest in both organisms during the infection. This network revealed a high degree of connectivity in many of the previously recognized pathways in Z. mays such as jasmonic acid, ethylene, and reactive oxygen species (ROS). For the pathogen A. flavus, a link between aflatoxin production and vesicular transport was identified within the network. There was significant interspecies correlation of expression between Z. mays and A. flavus for a subset of 104 Z. mays, and 1942 A. flavus genes. This resulted in an interspecies subnetwork enriched in multiple Z. mays genes involved in the production of ROS. In addition to the ROS from Z. mays, there was enrichment in the vesicular transport pathways and the aflatoxin pathway for A. flavus. Included in these genes, a key aflatoxin cluster regulator, AflS, was found to be co-regulated with multiple Z. mays ROS producing genes within the network, suggesting AflS may be monitoring host ROS levels. The entire GEN for both host and pathogen, and the subset of interspecies correlations, is presented as a tool for hypothesis generation and discovery for events in the early stages of fungal infection of Z. mays by A. flavus.

  12. Soluble polymorphic bank vole prion proteins induced by co-expression of quiescin sulfhydryl oxidase in E. coli and their aggregation behaviors.

    Science.gov (United States)

    Abskharon, Romany; Dang, Johnny; Elfarash, Ameer; Wang, Zerui; Shen, Pingping; Zou, Lewis S; Hassan, Sedky; Wang, Fei; Fujioka, Hisashi; Steyaert, Jan; Mulaj, Mentor; Surewicz, Witold K; Castilla, Joaquín; Wohlkonig, Alexandre; Zou, Wen-Quan

    2017-10-04

    The infectious prion protein (PrPSc or prion) is derived from its cellular form (PrPC) through a conformational transition in animal and human prion diseases. Studies have shown that the interspecies conversion of PrPC to PrPSc is largely swayed by species barriers, which is mainly deciphered by the sequence and conformation of the proteins among species. However, the bank vole PrPC (BVPrP) is highly susceptible to PrPSc from different species. Transgenic mice expressing BVPrP with the polymorphic isoleucine (109I) but methionine (109M) at residue 109 spontaneously develop prion disease. To explore the mechanism underlying the unique susceptibility and convertibility, we generated soluble BVPrP by co-expression of BVPrP with Quiescin sulfhydryl oxidase (QSOX) in Escherichia coli. Interestingly, rBVPrP-109M and rBVPrP-109I exhibited distinct seeded aggregation pathways and aggregate morphologies upon seeding of mouse recombinant PrP fibrils, as monitored by thioflavin T fluorescence and electron microscopy. Moreover, they displayed different aggregation behaviors induced by seeding of hamster and mouse prion strains under real-time quaking-induced conversion. Our results suggest that QSOX facilitates the formation of soluble prion protein and provide further evidence that the polymorphism at residue 109 of QSOX-induced BVPrP may be a determinant in mediating its distinct convertibility and susceptibility.

  13. AAV1 mediated co-expression of formylglycine-generating enzyme and arylsulfatase a efficiently corrects sulfatide storage in a mouse model of metachromatic leukodystrophy.

    Science.gov (United States)

    Kurai, Toshiyuki; Hisayasu, Sanae; Kitagawa, Ryo; Migita, Makoto; Suzuki, Hidenori; Hirai, Yukihiko; Shimada, Takashi

    2007-01-01

    Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA) and is characterized by deposition of sulfatide in all organs, particularly the nervous system. Recently, formylglycine-generating enzyme (FGE) was found to be essential for activation of sulfatases. This study examined the utility of FGE co-expression in AAV type 1 vector (AAV1)-mediated gene therapy of ASA knockout (MLD) mice. AAV1-ASA alone or AAV1-ASA and AAV1-FGE were co-injected into a single site of the hippocampus. Enzyme assay and immunohistochemical analysis showed that ASA was detected not only in the injected hemisphere but also in the non-injected hemisphere by 7 months after injection. Level of ASA activity and extent of ASA distribution were significantly enhanced by co-introduction of AAV1-FGE. Marked reductions in sulfatide levels were observed throughout the entire brain. The unexpectedly widespread distribution of ASA may be due to a combination of diffusion in extracellular spaces, transport through axons, and circulation in cerebrospinal fluid. The rotarod test revealed improvement of neurological functions. These results demonstrate that direct injection of AAV1 vectors expressing ASA and FGE represents a highly promising approach with significant implications for the development of clinical protocols for MLD gene therapy.

  14. Co-expression of Interleukin-15 Enhances the Protective Immune Responses Induced by Immunization with a Murine Malaria MVA-Based Vaccine Encoding the Circumsporozoite Protein.

    Science.gov (United States)

    Parra, Marcela; Liu, Xia; Derrick, Steven C; Yang, Amy; Molina-Cruz, Alvaro; Barillas-Mury, Carolina; Zheng, Hong; Thao Pham, Phuong; Sedegah, Martha; Belmonte, Arnel; Litilit, Dianne D; Waldmann, Thomas A; Kumar, Sanjai; Morris, Sheldon L; Perera, Liyanage P

    2015-01-01

    Malaria remains a major global public health problem with an estimated 200 million cases detected in 2012. Although the most advanced candidate malaria vaccine (RTS,S) has shown promise in clinical trials, its modest efficacy and durability have created uncertainty about the impact of RTS,S immunization (when used alone) on global malaria transmission. Here we describe the development and characterization of a novel modified vaccinia virus Ankara (MVA)-based malaria vaccine which co-expresses the Plasmodium yoelii circumsporozoite protein (CSP) and IL-15. Vaccination/challenge studies showed that C57BL/6 mice immunized with the MVA-CSP/IL15 vaccine were protected significantly better against a P. yoelii 17XNL sporozoite challenge than either mice immunized with an MVA vaccine expressing only CSP or naïve controls. Importantly, the levels of total anti-CSP IgG were elevated about 100-fold for the MVA-CSP/IL15 immunized group compared to mice immunized with the MVA-CSP construct that does not express IL-15. Among the IgG subtypes, the IL-15 expressing MVA-CSP vaccine induced levels of IgG1 (8 fold) and IgG2b (80 fold) higher than the MVA-CSP construct. The significantly enhanced humoral responses and protection detected after immunization with the MVA-CSP/IL15 vaccine suggest that this IL-15 expressing MVA construct could be considered in the development of future malaria immunization strategies.

  15. A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

    Science.gov (United States)

    Kalra, Rajkumar S.; Wadhwa, Renu

    2015-02-01

    Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing & transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.

  16. A gene co-expression network in whole blood of schizophrenia patients is independent of antipsychotic-use and enriched for brain-expressed genes.

    Directory of Open Access Journals (Sweden)

    Simone de Jong

    Full Text Available Despite large-scale genome-wide association studies (GWAS, the underlying genes for schizophrenia are largely unknown. Additional approaches are therefore required to identify the genetic background of this disorder. Here we report findings from a large gene expression study in peripheral blood of schizophrenia patients and controls. We applied a systems biology approach to genome-wide expression data from whole blood of 92 medicated and 29 antipsychotic-free schizophrenia patients and 118 healthy controls. We show that gene expression profiling in whole blood can identify twelve large gene co-expression modules associated with schizophrenia. Several of these disease related modules are likely to reflect expression changes due to antipsychotic medication. However, two of the disease modules could be replicated in an independent second data set involving antipsychotic-free patients and controls. One of these robustly defined disease modules is significantly enriched with brain-expressed genes and with genetic variants that were implicated in a GWAS study, which could imply a causal role in schizophrenia etiology. The most highly connected intramodular hub gene in this module (ABCF1, is located in, and regulated by the major histocompatibility (MHC complex, which is intriguing in light of the fact that common allelic variants from the MHC region have been implicated in schizophrenia. This suggests that the MHC increases schizophrenia susceptibility via altered gene expression of regulatory genes in this network.

  17. Lignin, mitochondrial family and photorespiratory transporter classification as case studies in using co-expression, co-response and protein locations to aid in identifying transport functions

    Directory of Open Access Journals (Sweden)

    Takayuki eTohge

    2014-03-01

    Full Text Available Whole genome sequencing and the relative ease of transcript profiling have facilitated the collection and data warehousing of immense quantities of expression data. However, a substantial proportion of genes are not yet functionally annotated a problem which is particularly acute for transport proteins. In Arabidopsis, for example, only a minor fraction of the estimated 700 intracellular transporters have been identified at the molecular genetic level. Furthermore it is only within the last couple of years that critical genes such as those encoding the final transport step required for the long distance transport of sucrose and the first transporter of the core photorespiratory pathway have been identified. Here we will describe how transcriptional coordination between genes of known function and non-annotated genes allows the identification of putative transporters on the premise that such co-expressed genes tend to be functionally related. We will additionally extend this to include the expansion of this approach to include phenotypic information from other levels of cellular organization such as proteomic and metabolomic data and provide case studies wherein this approach has successfully been used to fill knowledge gaps in important metabolic pathways and physiological processes.

  18. A Network Approach of Gene Co-expression in the Zea mays/Aspergillus flavus Pathosystem to Map Host/Pathogen Interaction Pathways

    Science.gov (United States)

    Musungu, Bryan M.; Bhatnagar, Deepak; Brown, Robert L.; Payne, Gary A.; OBrian, Greg; Fakhoury, Ahmad M.; Geisler, Matt

    2016-01-01

    A gene co-expression network (GEN) was generated using a dual RNA-seq study with the fungal pathogen Aspergillus flavus and its plant host Zea mays during the initial 3 days of infection. The analysis deciphered novel pathways and mapped genes of interest in both organisms during the infection. This network revealed a high degree of connectivity in many of the previously recognized pathways in Z. mays such as jasmonic acid, ethylene, and reactive oxygen species (ROS). For the pathogen A. flavus, a link between aflatoxin production and vesicular transport was identified within the network. There was significant interspecies correlation of expression between Z. mays and A. flavus for a subset of 104 Z. mays, and 1942 A. flavus genes. This resulted in an interspecies subnetwork enriched in multiple Z. mays genes involved in the production of ROS. In addition to the ROS from Z. mays, there was enrichment in the vesicular transport pathways and the aflatoxin pathway for A. flavus. Included in these genes, a key aflatoxin cluster regulator, AflS, was found to be co-regulated with multiple Z. mays ROS producing genes within the network, suggesting AflS may be monitoring host ROS levels. The entire GEN for both host and pathogen, and the subset of interspecies correlations, is presented as a tool for hypothesis generation and discovery for events in the early stages of fungal infection of Z. mays by A. flavus. PMID:27917194

  19. Co-Expression and Co-Localization of Cartilage Glycoproteins CHI3L1 and Lubricin in Osteoarthritic Cartilage: Morphological, Immunohistochemical and Gene Expression Profiles

    Science.gov (United States)

    Szychlinska, Marta Anna; Trovato, Francesca Maria; Di Rosa, Michelino; Malaguarnera, Lucia; Puzzo, Lidia; Leonardi, Rosy; Castrogiovanni, Paola; Musumeci, Giuseppe

    2016-01-01

    Osteoarthritis is the most common human arthritis characterized by degeneration of articular cartilage. Several studies reported that levels of human cartilage glycoprotein chitinase 3-like-1 (CHI3L1) are known as a potential marker for the activation of chondrocytes and the progression of Osteoarthritis (OA), whereas lubricin appears to be chondroprotective. The aim of this study was to investigate the co-expression and co-localization of CHI3L1 and lubricin in normal and osteoarthritic rat articular cartilage to correlate their modified expression to a specific grade of OA. Samples of normal and osteoarthritic rat articular cartilage were analyzed by the Kellgren–Lawrence OA severity scores, the Kraus’ modified Mankin score and the Histopathology Osteoarthritis Research Society International (OARSI) system for histomorphometric evaluations, and through CHI3L1 and lubricin gene expression, immunohistochemistry and double immuno-staining analysis. The immunoexpression and the mRNA levels of lubricin increased in normal cartilage and decreased in OA cartilage (normal vs. OA, p < 0.01). By contrast, the immunoexpression and the mRNA levels of CHI3L1 increased in OA cartilage and decreased in normal cartilage (normal vs. OA, p < 0.01). Our findings are consistent with reports suggesting that these two glycoproteins are functionally associated with the development of OA and in particular with grade 2/3 of OA, suggesting that in the future they could be helpful to stage the severity and progression of the disease. PMID:26978347

  20. Co-Expression and Co-Localization of Cartilage Glycoproteins CHI3L1 and Lubricin in Osteoarthritic Cartilage: Morphological, Immunohistochemical and Gene Expression Profiles

    Directory of Open Access Journals (Sweden)

    Marta Anna Szychlinska

    2016-03-01

    Full Text Available Osteoarthritis is the most common human arthritis characterized by degeneration of articular cartilage. Several studies reported that levels of human cartilage glycoprotein chitinase 3-like-1 (CHI3L1 are known as a potential marker for the activation of chondrocytes and the progression of Osteoarthritis (OA, whereas lubricin appears to be chondroprotective. The aim of this study was to investigate the co-expression and co-localization of CHI3L1 and lubricin in normal and osteoarthritic rat articular cartilage to correlate their modified expression to a specific grade of OA. Samples of normal and osteoarthritic rat articular cartilage were analyzed by the Kellgren–Lawrence OA severity scores, the Kraus’ modified Mankin score and the Histopathology Osteoarthritis Research Society International (OARSI system for histomorphometric evaluations, and through CHI3L1 and lubricin gene expression, immunohistochemistry and double immuno-staining analysis. The immunoexpression and the mRNA levels of lubricin increased in normal cartilage and decreased in OA cartilage (normal vs. OA, p < 0.01. By contrast, the immunoexpression and the mRNA levels of CHI3L1 increased in OA cartilage and decreased in normal cartilage (normal vs. OA, p < 0.01. Our findings are consistent with reports suggesting that these two glycoproteins are functionally associated with the development of OA and in particular with grade 2/3 of OA, suggesting that in the future they could be helpful to stage the severity and progression of the disease.

  1. Gene Co-Expression Analysis Inferring the Crosstalk of Ethylene and Gibberellin in Modulating the Transcriptional Acclimation of Cassava Root Growth in Different Seasons.

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    Treenut Saithong

    Full Text Available Cassava is a crop of hope for the 21st century. Great advantages of cassava over other crops are not only the capacity of carbohydrates, but it is also an easily grown crop with fast development. As a plant which is highly tolerant to a poor environment, cassava has been believed to own an effective acclimation process, an intelligent mechanism behind its survival and sustainability in a wide range of climates. Herein, we aimed to investigate the transcriptional regulation underlying the adaptive development of a cassava root to different seasonal cultivation climates. Gene co-expression analysis suggests that AP2-EREBP transcription factor (ERF1 orthologue (D142 played a pivotal role in regulating the cellular response to exposing to wet and dry seasons. The ERF shows crosstalk with gibberellin, via ent-Kaurene synthase (D106, in the transcriptional regulatory network that was proposed to modulate the downstream regulatory system through a distinct signaling mechanism. While sulfur assimilation is likely to be a signaling regulation for dry crop growth response, calmodulin-binding protein is responsible for regulation in the wet crop. With our initiative study, we hope that our findings will pave the way towards sustainability of cassava production under various kinds of stress considering the future global climate change.

  2. Gene co-expression network analysis identifies porcine genes associated with variation in metabolizing fenbendazole and flunixin meglumine in the liver.

    Science.gov (United States)

    Howard, Jeremy T; Ashwell, Melissa S; Baynes, Ronald E; Brooks, James D; Yeatts, James L; Maltecca, Christian

    2017-05-02

    Identifying individual genetic variation in drug metabolism pathways is of importance not only in livestock, but also in humans in order to provide the ultimate goal of giving the right drug at the right dose at the right time. Our objective was to identify individual genes and gene networks involved in metabolizing fenbendazole (FBZ) and flunixin meglumine (FLU) in swine liver. The population consisted of female and castrated male pigs that were sired by boars represented by 4 breeds. Progeny were randomly placed into groups: no drug (UNT), FLU or FBZ administered. Liver transcriptome profiles from 60 animals with extreme (i.e. fast or slow drug metabolism) pharmacokinetic (PK) profiles were generated from RNA sequencing. Multiple cytochrome P450 (CYP1A1, CYP2A19 and CYP2C36) genes displayed different transcript levels across treated versus UNT. Weighted gene co-expression network analysis identified 5 and 3 modules of genes correlated with PK parameters and a portion of these were enriched for biological processes relevant to drug metabolism for FBZ and FLU, respectively. Genes within identified modules were shown to have a higher transcript level relationship (i.e. connectivity) in treated versus UNT animals. Investigation into the identified genes would allow for greater insight into FBZ and FLU metabolism.

  3. Genome-wide analysis of cis-regulatory element structure and discovery of motif-driven gene co-expression networks in grapevine.

    Science.gov (United States)

    Wong, Darren Chern Jan; Lopez Gutierrez, Rodrigo; Gambetta, Gregory Alan; Castellarin, Simone Diego

    2017-06-01

    Coordinated transcriptional and metabolic reprogramming ensures a plant's continued growth and survival under adverse environmental conditions. Transcription factors (TFs) act to modulate gene expression through complex cis-regulatory element (CRE) interactions. Genome-wide analysis of known plant CREs was performed for all currently predicted protein-coding gene promoters in grapevine (Vitis vinifera L.). Many CREs such as abscisic acid (ABA)-responsive, drought-responsive, auxin-responsive, and evening elements, exhibit bona fide CRE properties such as strong position bias towards the transcription start site (TSS) and over-representation when compared with random promoters. Genes containing these CREs are enriched in a large repertoire of plant biological pathways. Large-scale transcriptome analyses also show that these CREs are highly implicated in grapevine development and stress response. Numerous CRE-driven modules in condition-specific gene co-expression networks (GCNs) were identified and many of these modules were highly enriched for plant biological functions. Several modules corroborate known roles of CREs in drought response, pathogen defense, cell wall metabolism, and fruit ripening, whereas others reveal novel functions in plants. Comparisons with Arabidopsis suggest a general conservation in promoter architecture, gene expression dynamics, and GCN structure across species. Systems analyses of CREs provide insights into the grapevine cis-regulatory code and establish a foundation for future genomic studies in grapevine. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  4. A Gene Co-Expression Network in Whole Blood of Schizophrenia Patients Is Independent of Antipsychotic-Use and Enriched for Brain-Expressed Genes

    Science.gov (United States)

    de Jong, Simone; Boks, Marco P. M.; Fuller, Tova F.; Strengman, Eric; Janson, Esther; de Kovel, Carolien G. F.; Ori, Anil P. S.; Vi, Nancy; Mulder, Flip; Blom, Jan Dirk; Glenthøj, Birte; Schubart, Chris D.; Cahn, Wiepke; Kahn, René S.; Horvath, Steve; Ophoff, Roel A.

    2012-01-01

    Despite large-scale genome-wide association studies (GWAS), the underlying genes for schizophrenia are largely unknown. Additional approaches are therefore required to identify the genetic background of this disorder. Here we report findings from a large gene expression study in peripheral blood of schizophrenia patients and controls. We applied a systems biology approach to genome-wide expression data from whole blood of 92 medicated and 29 antipsychotic-free schizophrenia patients and 118 healthy controls. We show that gene expression profiling in whole blood can identify twelve large gene co-expression modules associated with schizophrenia. Several of these disease related modules are likely to reflect expression changes due to antipsychotic medication. However, two of the disease modules could be replicated in an independent second data set involving antipsychotic-free patients and controls. One of these robustly defined disease modules is significantly enriched with brain-expressed genes and with genetic variants that were implicated in a GWAS study, which could imply a causal role in schizophrenia etiology. The most highly connected intramodular hub gene in this module (ABCF1), is located in, and regulated by the major histocompatibility (MHC) complex, which is intriguing in light of the fact that common allelic variants from the MHC region have been implicated in schizophrenia. This suggests that the MHC increases schizophrenia susceptibility via altered gene expression of regulatory genes in this network. PMID:22761806

  5. A novel subunit vaccine co-expressing GM-CSF and PCV2b Cap protein enhances protective immunity against porcine circovirus type 2 in piglets.

    Science.gov (United States)

    Zhang, Huawei; Qian, Ping; Peng, Bo; Shi, Lin; Chen, Huanchun; Li, Xiangmin

    2015-05-15

    Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated disease. Capsid (Cap) protein of PCV2 is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. GM-CSF is an immune adjuvant that enhances responses to vaccines. In this study, recombinant baculoviruses Ac-Cap and Ac-Cap-GM-CSF expressing the Cap protein alone and co-expressing the Cap protein and porcine GM-CSF, respectively, were constructed successfully. The target proteins were analyzed by western blotting and IFA. Further, these proteins were confirmed by electron microscopy, which showed that Cap proteins could self-assemble into virus-like particles having diameters of 17-25nm. Animal experiments showed that pigs immunized with Cap-GM-CSF subunit vaccine showed significantly higher levels of PCV2-specific antibodies and neutralizing antibodies than pigs immunized with the Cap subunit vaccine and a commercial vaccine (Ingelvac CircoFLEX; PGM-CSF subunit vaccine showed significantly higher average daily weight gain after wild-type PCV2 challenge than pigs receiving the other three vaccines (PGM-CSF was a powerful immunoadjuvant for PCV2 subunit vaccines because it enhanced humoral immune response and improved immune protection against PCV2 infection in pigs. Thus, the novel Cap-GM-CSF subunit vaccine has the potential to be used as an effective and safe vaccine candidate against PCV2 infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Co-expression of chimeric chitinase and a polygalacturonase-inhibiting protein in transgenic canola (Brassica napus) confers enhanced resistance to Sclerotinia sclerotiorum.

    Science.gov (United States)

    Ziaei, Mahboobeh; Motallebi, Mostafa; Zamani, Mohammad Reza; Panjeh, Nasim Zarin

    2016-06-01

    Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is one of the major fungal diseases of canola. To develop resistance against this fungal disease, the chit42 from Trichoderma atroviride with chitin-binding domain and polygalacturonase-inhibiting protein 2 (PG1P2) of Phaseolus vulgaris were co-expressed in canola via Agrobacterium-mediated transformation. Stable integration and expression of transgenes in T0 and T2 plants was confirmed by PCR, Southern blot and RT-PCR analyses. Chitinase activity and PGIP2 inhibition were detected by colorimetric and agarose diffusion assay in transgenic lines but not in untransformed plants. The crude proteins from single copy transformant leaves having high chitinase and PGIP2 activity (T16, T8 and T3), showed up to 44 % inhibition of S. sclerotiorum hyphal growth. The homozygous T2 plants, showing inheritance in Mendelian fashion (3:1), were further evaluated under greenhouse conditions for resistance to S. sclerotiorum. Intact plants contaminated with mycelia showed resistance through delayed onset of the disease and restricted size and expansion of lesions as compared to wild type plants. Combined expression of chimeric chit42 and pgip2 in Brassica napus L. provide subsequent protection against SSR disease and can be helpful in increasing the canola production in Iran.

  7. Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies

    Science.gov (United States)

    Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Zhang, Kai; Batt, Micheal; Fitchett, Jonathan R; He, Dongmei; Rick, Heather L; Conner, Elaine M; Demarest, Stephen J

    2015-01-01

    Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the α/β TCR. Replacing CH1 with Cα and CL with Cβ, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of Cβ with a canonical β-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the CH1/CL and Cα/Cβ domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases. PMID:25611120

  8. NS1643 enhances ionic currents in a G604S-WT hERG co-expression system associated with long QT syndrome 2.

    Science.gov (United States)

    Huo, JianHua; Guo, Xueyan; Lu, Qun; Qiang, Hua; Liu, Ping; Bai, Ling; Huang, Christopher Lh; Zhang, Yanmin; Ma, Aiqun

    2017-11-01

    Loss of function mutations in the human ether-a-go-go-related gene (hERG) cause long QT syndrome type 2 (LQT2). Most LQT2 patients are heterozygous mutation carriers in which the mutant hERG exerts potent dominant-negative effects. 1, 3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) is known to enhance IKr in WT-hERG. We investigated its actions following lipofectamine-induced expression of both mutant G604S- and WT-hERG in the heterologous HEK293 expression system. Cells transfected with pcDNA3-G604S-hERG did not lead to any expression of detectable currents whether before or following NS1643 challenge. Cells transfected with both pcDNA3-WT-hERG and pcDNA3-G604S-hERG showed reduced hERG currents compared to those transfected with pcDNA3-G604S-hERG consistent with the reduced trafficking and formation of modified heteromeric WT-G604S channels reported on earlier occasions. Nevertheless, NS1643 then continued to produce concentration- and voltage-dependent increases in hERG current amplitude. It did not affect the voltage dependence of activation, recovery from inactivation and deactivation. However, NS1643 (30 μmol/L) slowed steady state inactivation and shifted the steady state half maximal activation voltage (V1/2 ) of the inactivation curve by +10 mV, and significantly increased the time constants of inactivation. Our present experimental results suggest that NS1643 significantly increases ion current and attenuates its inactivation in cells co-expressing G604S-hERG and WT-hERG. These findings raise the possibility that hERG channel activators offer potential treatment strategies for inherited LQT2. © 2017 John Wiley & Sons Australia, Ltd.

  9. Low co-expression of epidermal growth factor receptor and its chaperone heat shock protein 90 is associated with worse prognosis in primary glioblastoma, IDH-wild-type.

    Science.gov (United States)

    Sartori, Elsa; Langer, Rupert; Vassella, Erik; Hewer, Ekkehard; Schucht, Philippe; Zlobec, Inti; Berezowska, Sabina

    2017-10-01

    Epidermal growth factor receptor (EGFR) is a major oncogenic driver in glioblastoma (GBM) without mutations in the isocitrate dehydrogenase gene (IDH-wildtype). Heat shock protein 90 (HSP90) is a regulator of the stability of oncogenic proteins including EGFR, thereby acting as a molecular chaperone. We investigated the expression of EGFR and its chaperone HSP90 in GBM, IDH-wildtype. Tissue availability permitted analysis of 237/449 consecutive GBM cases, among them 214 IDH-wildtype (90.3%). The expression of EGFR and HSP90 was analysed by immunohistochemistry on a tissue microarray containing various tumour regions. The expression intensity (EI), and an expression score (ES) combining the percentage of stained cells with EI were determined for both markers. Overall, there was a positive correlation between EGFR and HSP90 expression (EI; r=0.275, PIDH-wildtype cases, and high expression of EGFR (ES only) was in trend associated with better outcome, but failed to meet statyistical significance (P=0.061). A combination of EGFR and HSP90, however, discriminated between different prognostic groups, with EGFRlow/HSP90low tumours showing the worst prognosis in univariate analysis (P=0.001), and in multivariate analysis including the other relevant prognostic factors age, MGMT status and postoperative treatment [n=76; hazard ratio (HR)=0.571; 95% confidence interval (CI) 0.328-0.996; P=0.048]. EGFR expression stratified most pronounced among HSP90low tumours, where the EGFRhigh phenotype was associated with longer survival. Our results reveal a variable reliance on the signalling pathway by EGFR in GBM, IDH-wildtype. Low co-expression was associated with worse prognosis.

  10. CoLIde: a bioinformatics tool for CO-expression-based small RNA Loci Identification using high-throughput sequencing data.

    Science.gov (United States)

    Mohorianu, Irina; Stocks, Matthew Benedict; Wood, John; Dalmay, Tamas; Moulton, Vincent

    2013-07-01

    Small RNAs (sRNAs) are 20-25 nt non-coding RNAs that act as guides for the highly sequence-specific regulatory mechanism known as RNA silencing. Due to the recent increase in sequencing depth, a highly complex and diverse population of sRNAs in both plants and animals has been revealed. However, the exponential increase in sequencing data has also made the identification of individual sRNA transcripts corresponding to biological units (sRNA loci) more challenging when based exclusively on the genomic location of the constituent sRNAs, hindering existing approaches to identify sRNA loci. To infer the location of significant biological units, we propose an approach for sRNA loci detection called CoLIde (Co-expression based sRNA Loci Identification) that combines genomic location with the analysis of other information such as variation in expression levels (expression pattern) and size class distribution. For CoLIde, we define a locus as a union of regions sharing the same pattern and located in close proximity on the genome. Biological relevance, detected through the analysis of size class distribution, is also calculated for each locus. CoLIde can be applied on ordered (e.g., time-dependent) or un-ordered (e.g., organ, mutant) series of samples both with or without biological/technical replicates. The method reliably identifies known types of loci and shows improved performance on sequencing data from both plants (e.g., A. thaliana, S. lycopersicum) and animals (e.g., D. melanogaster) when compared with existing locus detection techniques. CoLIde is available for use within the UEA Small RNA Workbench which can be downloaded from: http://srna-workbench.cmp.uea.ac.uk.

  11. Functional differentiation and spatial-temporal co-expression networks of the NBS-encoding gene family in Jilin ginseng, Panax ginseng C.A. Meyer.

    Science.gov (United States)

    Yin, Rui; Zhao, Mingzhu; Wang, Kangyu; Lin, Yanping; Wang, Yanfang; Sun, Chunyu; Wang, Yi; Zhang, Meiping

    2017-01-01

    Ginseng, Panax ginseng C.A. Meyer, is one of the most important medicinal plants for human health and medicine. It has been documented that over 80% of genes conferring resistance to bacteria, viruses, fungi and nematodes are contributed by the nucleotide binding site (NBS)-encoding gene family. Therefore, identification and characterization of NBS genes expressed in ginseng are paramount to its genetic improvement and breeding. However, little is known about the NBS-encoding genes in ginseng. Here we report genome-wide identification and systems analysis of the NBS genes actively expressed in ginseng (PgNBS genes). Four hundred twelve PgNBS gene transcripts, derived from 284 gene models, were identified from the transcriptomes of 14 ginseng tissues. These genes were classified into eight types, including TNL, TN, CNL, CN, NL, N, RPW8-NL and RPW8-N. Seven conserved motifs were identified in both the Toll/interleukine-1 receptor (TIR) and coiled-coil (CC) typed genes whereas six were identified in the RPW8 typed genes. Phylogenetic analysis showed that the PgNBS gene family is an ancient family, with a vast majority of its genes originated before ginseng originated. In spite of their belonging to a family, the PgNBS genes have functionally dramatically differentiated and been categorized into numerous functional categories. The expressions of the across tissues, different aged roots and the roots of different genotypes. However, they are coordinating in expression, forming a single co-expression network. These results provide a deeper understanding of the origin, evolution and functional differentiation and expression dynamics of the NBS-encoding gene family in plants in general and in ginseng particularly, and a NBS gene toolkit useful for isolation and characterization of disease resistance genes and for enhanced disease resistance breeding in ginseng and related species.

  12. Pathways of Lipid Metabolism in Marine Algae, Co-Expression Network, Bottlenecks and Candidate Genes for Enhanced Production of EPA and DHA in Species of Chromista

    Directory of Open Access Journals (Sweden)

    Alice Mühlroth

    2013-11-01

    Full Text Available The importance of n-3 long chain polyunsaturated fatty acids (LC-PUFAs for human health has received more focus the last decades, and the global consumption of n-3 LC-PUFA has increased. Seafood, the natural n-3 LC-PUFA source, is harvested beyond a sustainable capacity, and it is therefore imperative to develop alternative n-3 LC-PUFA sources for both eicosapentaenoic acid (EPA, 20:5n-3 and docosahexaenoic acid (DHA, 22:6n-3. Genera of algae such as Nannochloropsis, Schizochytrium, Isochrysis and Phaedactylum within the kingdom Chromista have received attention due to their ability to produce n-3 LC-PUFAs. Knowledge of LC-PUFA synthesis and its regulation in algae at the molecular level is fragmentary and represents a bottleneck for attempts to enhance the n-3 LC-PUFA levels for industrial production. In the present review, Phaeodactylum tricornutum has been used to exemplify the synthesis and compartmentalization of n-3 LC-PUFAs. Based on recent transcriptome data a co-expression network of 106 genes involved in lipid metabolism has been created. Together with recent molecular biological and metabolic studies, a model pathway for n-3 LC-PUFA synthesis in P. tricornutum has been proposed, and is compared to industrialized species of Chromista. Limitations of the n-3 LC-PUFA synthesis by enzymes such as thioesterases, elongases, acyl-CoA synthetases and acyltransferases are discussed and metabolic bottlenecks are hypothesized such as the supply of the acetyl-CoA and NADPH. A future industrialization will depend on optimization of chemical compositions and increased biomass production, which can be achieved by exploitation of the physiological potential, by selective breeding and by genetic engineering.

  13. Prediction of operon-like gene clusters in the Arabidopsis thaliana genome based on co-expression analysis of neighboring genes.

    Science.gov (United States)

    Wada, Masayoshi; Takahashi, Hiroki; Altaf-Ul-Amin, Md; Nakamura, Kensuke; Hirai, Masami Y; Ohta, Daisaku; Kanaya, Shigehiko

    2012-07-15

    Operon-like arrangements of genes occur in eukaryotes ranging from yeasts and filamentous fungi to nematodes, plants, and mammals. In plants, several examples of operon-like gene clusters involved in metabolic pathways have recently been characterized, e.g. the cyclic hydroxamic acid pathways in maize, the avenacin biosynthesis gene clusters in oat, the thalianol pathway in Arabidopsis thaliana, and the diterpenoid momilactone cluster in rice. Such operon-like gene clusters are defined by their co-regulation or neighboring positions within immediate vicinity of chromosomal regions. A comprehensive analysis of the expression of neighboring genes therefore accounts a crucial step to reveal the complete set of operon-like gene clusters within a genome. Genome-wide prediction of operon-like gene clusters should contribute to functional annotation efforts and provide novel insight into evolutionary aspects acquiring certain biological functions as well. We predicted co-expressed gene clusters by comparing the Pearson correlation coefficient of neighboring genes and randomly selected gene pairs, based on a statistical method that takes false discovery rate (FDR) into consideration for 1469 microarray gene expression datasets of A. thaliana. We estimated that A. thaliana contains 100 operon-like gene clusters in total. We predicted 34 statistically significant gene clusters consisting of 3 to 22 genes each, based on a stringent FDR threshold of 0.1. Functional relationships among genes in individual clusters were estimated by sequence similarity and functional annotation of genes. Duplicated gene pairs (determined based on BLAST with a cutoff of Emetabolism, containing P450 genes restricted to the Brassica family and predicted to be involved in secondary metabolism. Operon-like clusters tend to include genes encoding bio-machinery associated with ribosomes, the ubiquitin/proteasome system, secondary metabolic pathways, lipid and fatty-acid metabolism, and the lipid

  14. Co-expression of Arabidopsis NHX1 and bar Improves the Tolerance to Salinity, Oxidative Stress, and Herbicide in Transgenic Mungbean

    Directory of Open Access Journals (Sweden)

    Sanjeev Kumar

    2017-11-01

    Full Text Available Mungbean is an important pulse crop extensively cultivated in Southeast Asia for supply of easily digestible protein. Salinity severely limits the growth and productivity of mungbean, and weeding poses nutritional and disease constraints to mungbean cultivation. To pyramid both salt tolerance and protection against herbicide in mungbean, the AtNHX1 encoding tonoplast Na+/H+ antiporter from Arabidopsis, and bar gene associated with herbicide resistance were co-expressed through Agrobacterium-mediated transformation. Stress inducible expression of AtNHX1 significantly improved tolerance under salt stress to ionic, osmotic, and oxidative stresses in transgenic mungbean plants compared to the wild type (WT plants, whereas constitutive expression of bar provided resistance to herbicide. Compared to WT, transgenic mungbean plants grew better with higher plant height, foliage, dry mass and seed yield under high salt stress (200 mM NaCl in the greenhouse. The improved performance of transgenic plants under salt stress was associated with enhanced sequestration of Na+ in roots by vacuolar Na+/H+ antiporter and limited transport of toxic Na+ to shoots, possibly by restricting Na+ influx into shoots. Transgenic plants showed better intracellular ion homeostasis, osmoregulation, reduced cell membrane damage, improved photosynthetic capacity, and transpiration rate as compared to WT when subjected to salt stress. Reduction in hydrogen peroxide and oxygen radical production indicated enhanced protection of transgenic plants to both salt- and methyl vialogen (MV-induced oxidative stress. This study laid a firm foundation for improving mungbean yield in saline lands in Southeast Asia.

  15. The Brassica rapa FLC homologue FLC2 is a key regulator of flowering time, identified through transcriptional co-expression networks.

    Science.gov (United States)

    Xiao, Dong; Zhao, Jian J; Hou, Xi L; Basnet, Ram K; Carpio, Dunia P D; Zhang, Ning W; Bucher, Johan; Lin, Ke; Cheng, Feng; Wang, Xiao W; Bonnema, Guusje

    2013-11-01

    The role of many genes and interactions among genes involved in flowering time have been studied extensively in Arabidopsis, and the purpose of this study was to investigate how effectively results obtained with the model species Arabidopsis can be applied to the Brassicacea with often larger and more complex genomes. Brassica rapa represents a very close relative, with its triplicated genome, with subgenomes having evolved by genome fractionation. The question of whether this genome fractionation is a random process, or whether specific genes are preferentially retained, such as flowering time (Ft) genes that play a role in the extreme morphological variation within the B. rapa species (displayed by the diverse morphotypes), is addressed. Data are presented showing that indeed Ft genes are preferentially retained, so the next intriguing question is whether these different orthologues of Arabidopsis Ft genes play similar roles compared with Arabidopsis, and what is the role of these different orthologues in B. rapa. Using a genetical-genomics approach, co-location of flowering quantitative trait loci (QTLs) and expression QTLs (eQTLs) resulted in identification of candidate genes for flowering QTLs and visualization of co-expression networks of Ft genes and flowering time. A major flowering QTL on A02 at the BrFLC2 locus co-localized with cis eQTLs for BrFLC2, BrSSR1, and BrTCP11, and trans eQTLs for the photoperiod gene BrCO and two paralogues of the floral integrator genes BrSOC1 and BrFT. It is concluded that the BrFLC2 Ft gene is a major regulator of flowering time in the studied doubled haploid population.

  16. Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells.

    Science.gov (United States)

    Weng, Xiaodong; Kuang, Youlin; Liu, Xiuheng; Chen, Zhiyuan; Zhu, Hengcheng; Chen, Hui; Jiang, Botao; Shen, Hao

    2011-03-01

    Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMax™ Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P m4-1BBL induced higher T-cell proliferation (OD(450) = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

  17. Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

    Directory of Open Access Journals (Sweden)

    Xiaodong Weng

    2011-03-01

    Full Text Available Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12 in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA. Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4% and CD86 (80.13 ± 2.81%] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL and IL-12 (249.57 ± 12.51 pg/mL production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05 than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018, indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

  18. Construction of a bivalent DNA vaccine co-expressing S genes of transmissible gastroenteritis virus and porcine epidemic diarrhea virus delivered by attenuated Salmonella typhimurium.

    Science.gov (United States)

    Zhang, Yudi; Zhang, Xiaohui; Liao, Xiaodan; Huang, Xiaobo; Cao, Sanjie; Wen, Xintian; Wen, Yiping; Wu, Rui; Liu, Wumei

    2016-06-01

    Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) can cause severe diarrhea in newborn piglets and led to significant economic losses. The S proteins are the main structural proteins of PEDV and TGEV capable of inducing neutralizing antibodies in vivo. In this study, a DNA vaccine SL7207 (pVAXD-PS1-TS) co-expressing S proteins of TGEV and PEDV delivered by attenuated Salmonella typhimurium was constructed and its immunogenicity in piglets was investigated. Twenty-day-old piglets were orally immunized with SL7207 (pVAXD-PS1-TS) at a dosage of 1.6 × 10(11) CFU per piglet and then booster immunized with 2.0 × 10(11) CFU after 2 weeks. Humoral immune responses, as reflected by virus neutralizing antibodies and specific IgG and sIgA, and cellular immune responses, as reflected by IFN-γ, IL-4, and lymphocyte proliferation, were evaluated. SL7207 (pVAXD-PS1-TS) simultaneously elicited immune responses against TGEV and PEDV after oral immunization. The immune levels started to increase at 2 weeks after immunization and increased to levels statistically significantly different than controls at 4 weeks post-immunization, peaking at 6 weeks and declined at 8 weeks. The humoral, mucosal, and cellular immune responses induced by SL7207 (pAXD-PS1-TS) were significantly higher than those of the PBS and SL7207 (pVAXD) (p < 0.01). In particular, the levels of IFN-γ and IL-4 were higher than those induced by the single-gene vaccine SL7207 (pVAXD-PS1) (p < 0.05). These results demonstrated that SL7207 (pVAXD-PS1-TS) possess the immunological functions of the two S proteins of TGEV and PEDV, indicating that SL7207 (pVAXD-PS1-TS) is a candidate oral vaccine for TGE and PED.

  19. A Genome-Wide Association Study for Culm Cellulose Content in Barley Reveals Candidate Genes Co-Expressed with Members of the CELLULOSE SYNTHASE A Gene Family

    Science.gov (United States)

    Houston, Kelly; Burton, Rachel A.; Sznajder, Beata; Rafalski, Antoni J.; Dhugga, Kanwarpal S.; Mather, Diane E.; Taylor, Jillian; Steffenson, Brian J.; Waugh, Robbie; Fincher, Geoffrey B.

    2015-01-01

    Cellulose is a fundamentally important component of cell walls of higher plants. It provides a scaffold that allows the development and growth of the plant to occur in an ordered fashion. Cellulose also provides mechanical strength, which is crucial for both normal development and to enable the plant to withstand both abiotic and biotic stresses. We quantified the cellulose concentration in the culm of 288 two – rowed and 288 six – rowed spring type barley accessions that were part of the USDA funded barley Coordinated Agricultural Project (CAP) program in the USA. When the population structure of these accessions was analysed we identified six distinct populations, four of which we considered to be comprised of a sufficient number of accessions to be suitable for genome-wide association studies (GWAS). These lines had been genotyped with 3072 SNPs so we combined the trait and genetic data to carry out GWAS. The analysis allowed us to identify regions of the genome containing significant associations between molecular markers and cellulose concentration data, including one region cross-validated in multiple populations. To identify candidate genes we assembled the gene content of these regions and used these to query a comprehensive RNA-seq based gene expression atlas. This provided us with gene annotations and associated expression data across multiple tissues, which allowed us to formulate a supported list of candidate genes that regulate cellulose biosynthesis. Several regions identified by our analysis contain genes that are co-expressed with CELLULOSE SYNTHASE A (HvCesA) across a range of tissues and developmental stages. These genes are involved in both primary and secondary cell wall development. In addition, genes that have been previously linked with cellulose synthesis by biochemical methods, such as HvCOBRA, a gene of unknown function, were also associated with cellulose levels in the association panel. Our analyses provide new insights into the

  20. Exposure to depleted uranium does not alter the co-expression of HER-2/neu and p53 in breast cancer patients

    Directory of Open Access Journals (Sweden)

    Al-Toriahi Kaswer M

    2011-03-01

    Full Text Available Abstract Background Amongst the extensive literature on immunohistochemical profile of breast cancer, very little is found on populations exposed to a potential risk factor such as depleted uranium. This study looked at the immunohistochemical expression of HER-2/neu (c-erbB2 and p53 in different histological types of breast cancer found in the middle Euphrates region of Iraq, where the population has been exposed to high levels of depleted uranium. Findings The present investigation was performed over a period starting from September 2008 to April 2009. Formalin-fixed, paraffin-embedded blocks from 70 patients with breast cancer (62 ductal and 8 lobular carcinoma were included in this study. A group of 25 patients with fibroadenoma was included as a comparative group, and 20 samples of normal breast tissue sections were used as controls. Labeled streptavidin-biotin (LSAB+ complex method was employed for immunohistochemical detection of HER-2/neu and p53. The detection rate of HER-2/neu and p53 immunohistochemical expression were 47.14% and 35.71% respectively in malignant tumors; expression was negative in the comparative and control groups (p HER-2/neu immunostaining was significantly associated with histological type, tumor size, nodal involvement, and recurrence of breast carcinoma (p p Both biomarkers were positively correlated with each other. Furthermore, all the cases that co-expressed both HER-2/neu and p53 showed the most unfavorable biopathological profile. Conclusion P53 and HER-2/neu over-expression play an important role in pathogenesis of breast carcinoma. The findings indicate that in regions exposed to high levels of depleted uranium, although p53 and HER-2/neu overexpression are both high, correlation of their expression with age, grade, tumor size, recurrence and lymph node involvement is similar to studies that have been conducted on populations not exposed to depleted uranium. HER-2/neu expression in breast cancer was higher

  1. miR-363-5p as potential prognostic marker for hepatocellular carcinoma indicated by weighted co-expression network analysis of miRNAs and mRNA

    OpenAIRE

    Zhang, Jun; Fan, Jia; Zhou, Chongming; Qi, Yanyu

    2017-01-01

    Background This study aimed to investigate potential miRNAs and genes associated with the prognosis of hepatocellular carcinoma (HCC). Methods Weighted co-expression network analysis was utilized to analyze the mRNA and miRNA sequencing data of HCC from TCGA (The Cancer Genome Atlas) database. Significant network modules were identified, and then functions of genes in the gene network modules and target genes of miRNAs in the miRNA network modules were explored. Additionally, correlations bet...

  2. Copper delivery to the CNS by CuATSM effectively treats motor neuron disease in SOD(G93A) mice co-expressing the Copper-Chaperone-for-SOD.

    Science.gov (United States)

    Williams, Jared R; Trias, Emiliano; Beilby, Pamela R; Lopez, Nathan I; Labut, Edwin M; Bradford, C Samuel; Roberts, Blaine R; McAllum, Erin J; Crouch, Peter J; Rhoads, Timothy W; Pereira, Cliff; Son, Marjatta; Elliott, Jeffrey L; Franco, Maria Clara; Estévez, Alvaro G; Barbeito, Luis; Beckman, Joseph S

    2016-05-01

    Over-expression of mutant copper, zinc superoxide dismutase (SOD) in mice induces ALS and has become the most widely used model of neurodegeneration. However, no pharmaceutical agent in 20 years has extended lifespan by more than a few weeks. The Copper-Chaperone-for-SOD (CCS) protein completes the maturation of SOD by inserting copper, but paradoxically human CCS causes mice co-expressing mutant SOD to die within two weeks of birth. Hypothesizing that co-expression of CCS created copper deficiency in spinal cord, we treated these pups with the PET-imaging agent CuATSM, which is known to deliver copper into the CNS within minutes. CuATSM prevented the early mortality of CCSxSOD mice, while markedly increasing Cu, Zn SOD protein in their ventral spinal cord. Remarkably, continued treatment with CuATSM extended the survival of these mice by an average of 18 months. When CuATSM treatment was stopped, these mice developed ALS-related symptoms and died within 3 months. Restoring CuATSM treatment could rescue these mice after they became symptomatic, providing a means to start and stop disease progression. All ALS patients also express human CCS, raising the hope that familial SOD ALS patients could respond to CuATSM treatment similarly to the CCSxSOD mice. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  3. In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus.

    Science.gov (United States)

    Mamedov, Tarlan; Cicek, Kader; Gulec, Burcu; Ungor, Rifat; Hasanova, Gulnara

    2017-01-01

    A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-β-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes.

  4. In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H of Streptomyces plicatus.

    Directory of Open Access Journals (Sweden)

    Tarlan Mamedov

    Full Text Available A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F. Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-β-N-acetylglucosaminidase (EC3.2.1.96, Endo H, another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes.

  5. Identification of duplicated and stress-inducible Aox2b gene co-expressed with Aox1 in species of the Medicago genus reveals a regulation linked to gene rearrangement in leguminous genomes.

    Science.gov (United States)

    Cavalcanti, João Henrique Frota; Oliveira, Georgia Mesquita; Saraiva, Kátia Daniella da Cruz; Torquato, José Pedro Pires; Maia, Ivan G; de Melo, Dirce Fernandes; Costa, José Hélio

    2013-12-15

    In flowering plants, alternative oxidase (Aox) is encoded by 3-5 genes distributed in 2 subfamilies (Aox1 and Aox2). In several species only Aox1 is reported as a stress-responsive gene, but in the leguminous Vigna unguiculata Aox2b is also induced by stress. In this work we investigated the Aox genes from two leguminous species of the Medicago genus (Medicago sativa and Medicago truncatula) which present one Aox1, one Aox2a and an Aox2b duplication (named here Aox2b1 and Aox2b2). Expression analyses by semi-quantitative RT-PCR in M. sativa revealed that Aox1, Aox2b1 and Aox2b2 transcripts increased during seed germination. Similar analyses in leaves and roots under different treatments (SA, PEG, H2O2 and cysteine) revealed that these genes are also induced by stress, but with peculiar spatio-temporal differences. Aox1 and Aox2b1 showed basal levels of expression under control conditions and were induced by stress in leaves and roots. Aox2b2 presented a dual behavior, i.e., it was expressed only under stress conditions in leaves, and showed basal expression levels in roots that were induced by stress. Moreover, Aox2a was expressed at higher levels in leaves and during seed germination than in roots and appeared to be not responsive to stress. The Aox expression profiles obtained from a M. truncatula microarray dataset also revealed a stress-induced co-expression of Aox1, Aox2b1 and Aox2b2 in leaves and roots. These results reinforce the stress-inducible co-expression of Aox1/Aox2b in some leguminous plants. Comparative genomic analysis indicates that this regulation is linked to Aox1/Aox2b proximity in the genome as a result of the gene rearrangement that occurred in some leguminous plants during evolution. The differential expression of Aox2b1/2b2 suggests that a second gene has been originated by recent gene duplication with neofunctionalization. Copyright © 2013 Elsevier GmbH. All rights reserved.

  6. Prognostic value of the MicroRNA regulators Dicer and Drosha in non-small-cell lung cancer: co-expression of Drosha and miR-126 predicts poor survival.

    Science.gov (United States)

    Lønvik, Kenneth; Sørbye, Sveinung W; Nilsen, Marit N; Paulssen, Ruth H

    2014-01-01

    Dicer and Drosha are important enzymes for processing microRNAs. Recent studies have exhibited possible links between expression of different miRNAs, levels of miRNA processing enzymes, and cancer prognosis. We have investigated the prognostic impact of Dicer and Drosha and their correlation with miR-126 expression in a large cohort of non-small cell lung cancer (NSCLC) patients. We aimed to find patient groups within the cohort that might have an advantage of receiving adjunctive therapies. Dicer expression in the cytoplasm and Drosha expression in the nucleus were evaluated by manual immunohistochemistry of tissue microarrays (TMAs), including tumor tissue samples from 335 patients with resected stages I to IIIA NSCLC. In addition, in situ hybridizations of TMAs for visualization of miR-126 were performed. Kaplan-Meier analysis was performed, and the log-rank test via SPSS v.22 was used for estimating significance levels. In patients with normal performance status (ECOG = 0, n = 197), high Dicer expression entailed a significantly better prognosis than low Dicer expression (P = 0.024). Dicer had no significant prognostic value in patients with reduced performance status (ECOG = 1-2, n = 138). High Drosha expression was significantly correlated with high levels of the microRNA 126 (miR-126) (P = 0.004). Drosha/miR-126 co-expression had a significant negative impact on the disease-specific survival (DSS) rate (P < 0.001). Multivariate analyses revealed that the interaction Dicer*Histology (P = 0.049) and Drosha/miR-126 co-expression (P = 0.033) were independent prognostic factors. In NSCLC patients with normal performance status, Dicer is a positive prognostic factor. The importance of Drosha as a prognostic factor in our material seems to be related to miR-126 and possibly other microRNAs.

  7. Expression analysis of mammaglobin A (SCGB2A2 and lipophilin B (SCGB1D2 in more than 300 human tumors and matching normal tissues reveals their co-expression in gynecologic malignancies

    Directory of Open Access Journals (Sweden)

    Kristiansen Glen

    2006-04-01

    Full Text Available Abstract Background Mammaglobin A (SCGB2A2 and lipophilin B (SCGB1D2, two members of the secretoglobin superfamily, are known to be co-expressed in breast cancer, where their proteins form a covalent complex. Based on the relatively high tissue-specific expression pattern, it has been proposed that the mammaglobin A protein and/or its complex with lipophilin B could be used in breast cancer diagnosis and treatment. In view of these clinical implications, the aim of the present study was to analyze the expression of both genes in a large panel of human solid tumors (n = 309, corresponding normal tissues (n = 309 and cell lines (n = 11, in order to evaluate their tissue specific expression and co-expression pattern. Methods For gene and protein expression analyses, northern blot, dot blot hybridization of matched tumor/normal arrays (cancer profiling arrays, quantitative RT-PCR, non-radioisotopic RNA in situ hybridization and immunohistochemistry were used. Results Cancer profiling array data demonstrated that mammaglobin A and lipophilin B expression is not restricted to normal and malignant breast tissue. Both genes were abundantly expressed in tumors of the female genital tract, i.e. endometrial, ovarian and cervical cancer. In these four tissues the expression pattern of mammaglobin A and lipophilin B was highly concordant, with both genes being down-, up- or not regulated in the same tissue samples. In breast tissue, mammaglobin A expression was down-regulated in 49% and up-regulated in 12% of breast tumor specimens compared with matching normal tissues, while lipophilin B was down-regulated in 59% and up-regulated in 3% of cases. In endometrial tissue, expression of mammaglobin A and lipophilin B was clearly up-regulated in tumors (47% and 49% respectively. Both genes exhibited down-regulation in 22% of endometrial tumors. The only exceptions to this concordance of mammaglobin A/lipophilin B expression were normal and malignant tissues of

  8. Co-expression of cytokeratins and vimentin by highly invasive trophoblast in the white-winged vampire bat, Diaemus youngi, and the black mastiff bat, Molossus ater, with observations on intermediate filament proteins in the decidua and intraplacental trophoblast.

    Science.gov (United States)

    Badwaik, N K; Rasweiler, J J; Muradali, F

    1998-11-01

    Histological and immunocytochemical studies of gravid reproductive tracts obtained from the white-winged vampire bat (Diaemus youngi) and the black mastiff bat (Molossus ater) have established that both species develop unusually invasive trophoblast. This is released by the developing discoidal haemochorial placenta, expresses both cytokeratins and vimentin, and invades the myometrium and adjacent tissues (including the ovaries) via interstitial migration within the walls of maternal blood vessels. Hence, this trophoblast is noteworthy for the extent to which it undergoes an epithelial-mesenchymal transformation. In Molossus, it originates from the cytotrophoblastic shell running along the base of the placenta, is mononuclear, and preferentially invades maternal arterial vessels serving the discoidal placenta. This trophoblast may have a role in dilatation of these vessels when the discoidal placenta becomes functional. In Diaemus, the highly invasive trophoblast appears to originate instead from a layer of syncytiotrophoblast on the periphery of the placenta is multinucleated, and vigorously invades both arterial and venous vessels. During late pregnancy, it becomes extensively branched and sends attenuated processes around many of the myometrial smooth muscle fibres. In view of its distribution, this trophoblast could have important influences upon myometrial contractility and the function of blood vessels serving the gravid tract. Other aspects of intermediate filament expression in the uteri and placentae of these bats are also noteworthy. Many of the decidual giant cells in Molossus co-express cytokeratins and vimentin, while the syncytiotrophoblast lining the placental labyrinth in Diaemus late in pregnancy expresses little cytokeratin.

  9. Transgenic alfalfa plants co-expressing glutathione S-transferase (GST) and human CYP2E1 show enhanced resistance to mixed contaminates of heavy metals and organic pollutants

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yuanyuan [Department of Pharmaceutics, Qingdao University of Science and Technology, 53 Zhengzhou Road, P.O. Box 70, Qingdao 266042 (China); Liu, Junhong, E-mail: liujh@qust.edu.cn [Department of Pharmaceutics, Qingdao University of Science and Technology, 53 Zhengzhou Road, P.O. Box 70, Qingdao 266042 (China)

    2011-05-15

    Transgenic alfalfa plants simultaneously expressing human CYP2E1 and glutathione S-transferase (GST) were generated from hypocotyl segments by the use of an Agrobacterium transformation system for the phytoremediation of the mixed contaminated soil with heavy metals and organic pollutants. The transgenic alfalfa plants were screened by a combination of kanamycin resistance, PCR, GST and CYP2E1 activity and Western blot analysis. The capabilities of mixed contaminants (heavy metals-organic compounds) resistance of pKHCG transgenic alfalfa plants became markedly increased compared with the transgenic alfalfa plants expressing single gene (GST or CYP2E1) and the non-transgenic control plants. The pKHCG alfalfa plants exhibited strong resistance towards the mixtures of cadmium (Cd) and trichloroethylene (TCE) that were metabolized by the introduced GST and CYP2E1 in combination. Our results show that the pKHCG transgenic alfalfa plants have good potential for phytoremediation because they have cross-tolerance towards the complex contaminants of heavy metals and organic pollutants. Therefore, these transgenic alfalfa plants co-expressing GST and human P450 CDNAs may have a great potential for phytoremediation of mixed environmental contaminants.

  10. Transgenic alfalfa plants co-expressing glutathione S-transferase (GST) and human CYP2E1 show enhanced resistance to mixed contaminates of heavy metals and organic pollutants.

    Science.gov (United States)

    Zhang, Yuanyuan; Liu, Junhong

    2011-05-15

    Transgenic alfalfa plants simultaneously expressing human CYP2E1 and glutathione S-transferase (GST) were generated from hypocotyl segments by the use of an Agrobacterium transformation system for the phytoremediation of the mixed contaminated soil with heavy metals and organic pollutants. The transgenic alfalfa plants were screened by a combination of kanamycin resistance, PCR, GST and CYP2E1 activity and Western blot analysis. The capabilities of mixed contaminants (heavy metals-organic compounds) resistance of pKHCG transgenic alfalfa plants became markedly increased compared with the transgenic alfalfa plants expressing single gene (GST or CYP2E1) and the non-transgenic control plants. The pKHCG alfalfa plants exhibited strong resistance towards the mixtures of cadmium (Cd) and trichloroethylene (TCE) that were metabolized by the introduced GST and CYP2E1 in combination. Our results show that the pKHCG transgenic alfalfa plants have good potential for phytoremediation because they have cross-tolerance towards the complex contaminants of heavy metals and organic pollutants. Therefore, these transgenic alfalfa plants co-expressing GST and human P450 CDNAs may have a great potential for phytoremediation of mixed environmental contaminants. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Protection of guinea pigs by vaccination with a recombinant swinepox virus co-expressing HA1 genes of swine H1N1 and H3N2 influenza viruses.

    Science.gov (United States)

    Xu, Jiarong; Yang, Deji; Huang, Dongyan; Xu, Jiaping; Liu, Shichao; Lin, Huixing; Zhu, Haodan; Liu, Bao; Lu, Chengping

    2013-03-01

    Swine influenza (SI) is an acute respiratory infectious disease of swine caused by swine influenza virus (SIV). SIV is not only an important respiratory pathogen in pigs but also a potent threat to human health. Here, we report the construction of a recombinant swinepox virus (rSPV/H3-2A-H1) co-expressing hemagglutinin (HA1) of SIV subtypes H1N1 and H3N2. Immune responses and protection efficacy of the rSPV/H3-2A-H1 were evaluated in guinea pigs. Inoculation of rSPV/H3-2A-H1 yielded neutralizing antibodies against SIV H1N1 and H3N2. The IFN-γ and IL-4 concentrations in the supernatant of lymphocytes stimulated with purified SIV HA1 antigen were significantly higher (P guinea pigs against SIV H1N1 or H3N2 challenge was observed. No SIV shedding was detected from guinea pigs vaccinated with rSPV/H3-2A-H1 after challenge. Most importantly, the guinea pigs immunized with rSPV/H3-2A-H1 did not show gross and micrographic lung lesions. However, the control guinea pigs experienced distinct gross and micrographic lung lesions at 7 days post-challenge. Our data suggest that the recombinant swinepox virus encoding HA1 of SIV H1N1 and H3N2 might serve as a promising candidate vaccine for protection against SIV H1N1 and H3N2 infections.

  12. Analysis of cellulose synthase genes from domesticated apple identifies collinear genes WDR53 and CesA8A: partial co-expression, bicistronic mRNA, and alternative splicing of CESA8A.

    Science.gov (United States)

    Guerriero, Gea; Spadiut, Oliver; Kerschbamer, Christine; Giorno, Filomena; Baric, Sanja; Ezcurra, Inés

    2012-10-01

    Cellulose synthase (CesA) genes constitute a complex multigene family with six major phylogenetic clades in angiosperms. The recently sequenced genome of domestic apple, Malus×domestica, was mined for CesA genes, by blasting full-length cellulose synthase protein (CESA) sequences annotated in the apple genome against protein databases from the plant models Arabidopsis thaliana and Populus trichocarpa. Thirteen genes belonging to the six angiosperm CesA clades and coding for proteins with conserved residues typical of processive glycosyltransferases from family 2 were detected. Based on their phylogenetic relationship to Arabidopsis CESAs, as well as expression patterns, a nomenclature is proposed to facilitate further studies. Examination of their genomic organization revealed that MdCesA8-A is closely linked and co-oriented with WDR53, a gene coding for a WD40 repeat protein. The WDR53 and CesA8 genes display conserved collinearity in dicots and are partially co-expressed in the apple xylem. Interestingly, the presence of a bicistronic WDR53-CesA8A transcript was detected in phytoplasma-infected phloem tissues of apple. The bicistronic transcript contains a spliced intergenic sequence that is predicted to fold into hairpin structures typical of internal ribosome entry sites, suggesting its potential cap-independent translation. Surprisingly, the CesA8A cistron is alternatively spliced and lacks the zinc-binding domain. The possible roles of WDR53 and the alternatively spliced CESA8 variant during cellulose biosynthesis in M.×domestica are discussed.

  13. Evaluation of a DNA vaccine candidate co-expressing GP3 and GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) with interferon α/γ in immediate and long-lasting protection against HP-PRRSV challenge.

    Science.gov (United States)

    Du, Yijun; Qi, Jing; Lu, Yu; Wu, Jiaqiang; Yoo, Dongwan; Liu, Xing; Zhang, Xiumei; Li, Jun; Sun, Wenbo; Cong, Xiaoyan; Shi, Jianli; Wang, Jinbao

    2012-12-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) has become one of the most economically important diseases to the global pork industry. Current vaccination strategies only provide a limited protective efficacy. In this study, a DNA vaccine, pVAX1(©)-α-γ-GP35, co-expressing GP3 and GP5 of PRRSV with interferon α/γ was constructed, and its immediate and long-lasting protection against highly pathogenic PRRSV (HP-PRRSV) challenge were examined in pigs. For immediate protection, the results showed that pVAX1(©)-α-γ-GP35 could provide partially protective efficacy, which was similar to the pVAX1(©)-α-γ (expressing interferon α/γ). For long-lasting protection, pigs inoculated with pVAX1(©)-α-γ-GP35 developed significantly higher PRRSV-specific antibody response, T cell proliferation, IFN-γ, and IL-4, than those vaccinated with pVAX1(©)-GP35 (expressing GP3 and GP5 of PRRSV). Following homologous challenge with HP-PRRSV strain SD-JN, pigs inoculated with pVAX1(©)-α-γ-GP35 showed almost no clinical signs, no lung lesions, and significantly lower viremia, as compared to those in pVAX1(©)-GP35 group. It indicated that pVAX1(©)-α-γ-GP35 could induce enhanced immune responses and provide both immediate and long-lasting protection against HP-PRRSV challenge in pigs. The DNA vaccine pVAX1(©)-α-γ-GP35 might be an attractive candidate vaccine for the prevention and control of HP-PRRSV infections.

  14. Molecular Cloning, Structural Modeling and the Production of Soluble Triple-Mutated Diphtheria Toxoid (K51E/G52E/E148K) Co-expressed with Molecular Chaperones in Recombinant Escherichia coli.

    Science.gov (United States)

    Uthailak, Naphatsamon; Mahamad, Pornpimol; Chittavanich, Pamorn; Yanarojana, Somchai; Wijagkanalan, Wassana; Petre, Jean; Panbangred, Watanalai

    2017-05-01

    CRM197 is a diphtheria toxin (DT) mutant (G52E) which has been used as a carrier protein for conjugate vaccines. However, it still possesses cytotoxicity toward mammalian cells. The goal of this project was to produce a non-toxic and soluble CRM197EK through introduction of triple amino acid substitutions (K51E/G52E/E148K) in Escherichia coli. The expression of CRM197EKTrxHis was optimized and co-expressed with different molecular chaperones. The soluble CRM197EKTrxHis was produced at a high concentration (97.33 ± 17.47 μg/ml) under the optimal condition (induction with 0.1 mM IPTG at 20 °C for 24 h). Cells containing pG-Tf2, expressing trigger factor and GroEL-GroES, accumulated the highest amount of soluble CRM197EKTrxHis at 111.24 ± 10.40 μg/ml after induction for 24 h at 20 °C. The soluble CRM197EKTrxHis still possesses nuclease activity and completely digest λDNA at 25 and 37 °C with 8- and 4-h incubation, respectively. Molecular modeling of diphtheria toxin, CRM197 and CRM197EK indicated that substitutions of two amino acids (K51E/E148K) may cause poor NAD binding, consistent with the lack of toxicity. Therefore, CRM197EK might be used as a new potential carrier protein. However, further in vivo study is required to confirm its roles as functional carrier protein in conjugate vaccines.

  15. Redox proteomics analyses of the influence of co-expression of wild-type or mutated LRRK2 and Tau on C. elegans protein expression and oxidative modification: relevance to Parkinson disease.

    Science.gov (United States)

    Di Domenico, Fabio; Sultana, Rukhsana; Ferree, Andrew; Smith, Katelyn; Barone, Eugenio; Perluigi, Marzia; Coccia, Raffaella; Pierce, William; Cai, Jian; Mancuso, Cesare; Squillace, Rachel; Wiengele, Manfred; Dalle-Donne, Isabella; Wolozin, Benjamin; Butterfield, D Allan

    2012-12-01

    The human LRRK2 gene has been identified as the most common causative gene of autosomal-dominantly inherited and idiopathic Parkinson disease (PD). The G2019S substitution is the most common mutation in LRRK2. The R1441C mutation also occurs in cases of familial PD, but is not as prevalent. Some cases of LRRK2-based PD exhibit Tau pathology, which suggests that alterations on LRRK2 activity affect the pathophysiology of Tau. To investigate how LRRK2 might affect Tau and the pathophysiology of PD, we generated lines of C. elegans expressing human LRRK2 [wild-type (WT) or mutated (G2019S or R1441C)] with and without V337M Tau. Expression and redox proteomics were used to identify the effects of LRRK2 (WT and mutant) on protein expression and oxidative modifications. Co-expression of WT LRRK2 and Tau led to increased expression of numerous proteins, including several 60S ribosomal proteins, mitochondrial proteins, and the V-type proton ATPase, which is associated with autophagy. C. elegans expressing mutant LRRK2 showed similar changes, but also showed increased protein oxidation and lipid peroxidation, the latter indexed as increased protein-bound 4-hydroxy-2-nonenal (HNE). Our study brings new knowledge about the possible alterations induced by LRRK2 (WT and mutated) and Tau interactions, suggesting the involvement of G2019S and R1441C in Tau-dependent neurodegenerative processes. These results suggest that changes in LRRK2 expression or activity lead to corresponding changes in mitochondrial function, autophagy, and protein translation. These findings are discussed with reference to the pathophysiology of PD.

  16. Development of Caco-2 cells co-expressing CYP3A4 and NADPH-cytochrome P450 reductase using a human artificial chromosome for the prediction of intestinal extraction ratio of CYP3A4 substrates.

    Science.gov (United States)

    Takenaka, Toru; Kazuki, Kanako; Harada, Naomoto; Kuze, Jiro; Chiba, Masato; Iwao, Takahiro; Matsunaga, Tamihide; Abe, Satoshi; Oshimura, Mitsuo; Kazuki, Yasuhiro

    2017-02-01

    The Caco-2 cells co-expressing cytochrome P450 (CYP) 3A4 and NADPH-cytochrome P450 reductase (CPR) were developed using a human artificial chromosome (HAC) vector. The CYP3A4 and CPR genes were cloned into the HAC vector in CHO cells using the Cre-loxP system, and the microcell-mediated chromosome transfer technique was used to transfer the CYP3A4-CPR-HAC vector to Caco-2 cells. After seeding onto semipermeable culture inserts, the CYP3A4-CPR-HAC/Caco-2 cells were found to form tight monolayers, similar to the parental cells, as demonstrated by the high transepithelial electrical resistance (TEER) value and comparable permeability of non-CYP3A4 substrates between parent and CYP3A4-CPR-HAC/Caco-2 cell monolayers. The metabolic activity of CYP3A4 (midazolam 1'-hydroxylase activity) in the CYP3A4-CPR-HAC/Caco-2 cells was constant from 22 to 35 passages, indicating that HAC vectors conferred sufficient and sustained CYP3A4 activity to CYP3A4-CPR-HAC/Caco-2 cells. The strong relationship between the metabolic extraction ratios (ER) obtained from the CYP3A4-CPR-HAC/Caco-2 cells and calculated intestinal extraction ratios in humans (Eg) from reported intestinal availability (Fg) was found for 17 substrates of CYP3A4 (r2 = 0.84). The present study suggests that the CYP3A4-CPR-HAC/Caco-2 cell monolayer can serve as an in vitro tool that facilitates the prediction of intestinal extraction ratio (or availability) in humans. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  17. Ethanol production from acid- and alkali-pretreated corncob by endoglucanase and β-glucosidase co-expressing Saccharomyces cerevisiae subject to the expression of heterologous genes and nutrition added.

    Science.gov (United States)

    Feng, Chunying; Zou, Shaolan; Liu, Cheng; Yang, Huajun; Zhang, Kun; Ma, Yuanyuan; Hong, Jiefang; Zhang, Minhua

    2016-05-01

    Low-cost technologies to overcome the recalcitrance of cellulose are the key to widespread utilization of lignocellulosic biomass for ethanol production. Efficient enzymatic hydrolysis of cellulose requires the synergism of various cellulases, and the ratios of each cellulase are required to be regulated to achieve the maximum hydrolysis. On the other hand, engineering of cellulolytic Saccharomyces cerevisiae strains is a promising strategy for lignocellulosic ethanol production. The expression of cellulase-encoding genes in yeast would affect the synergism of cellulases and thus the fermentation ability of strains with exogenous enzyme addition. However, such researches are rarely reported. In this study, ten endoglucanase and β-glucosidase co-expressing S. cerevisiae strains were constructed and evaluated by enzyme assay and fermentation performance measurement. The results showed that: (1) maximum ethanol titers of recombinant strains exhibited high variability in YPSC medium (20 g/l peptone, 10 g/l yeast extract, 100 g/l acid- and alkali-pretreated corncob) within 10 days. However, they had relatively little difference in USC medium (100 g/l acid- and alkali-pretreated corncob, 0.33 g/l urea, pH 5.0). (2) Strains 17# and 19#, with ratio (CMCase to β-glucosidase) of 7.04 ± 0.61 and 7.40 ± 0.71 respectively, had the highest fermentation performance in YPSC. However, strains 11# and 3# with the highest titers in USC medium had a higher ratio of CMCase to β-glucosidase, and CMCase activities. These results indicated that nutrition, enzyme activities and the ratio of heterologous enzymes had notable influence on the fermentation ability of cellulase-expressing yeast.

  18. Efficient whole-cell biocatalyst for acetoin production with NAD+ regeneration system through homologous co-expression of 2,3-butanediol dehydrogenase and NADH oxidase in engineered Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Teng Bao

    Full Text Available Acetoin (3-hydroxy-2-butanone, an extensively-used food spice and bio-based platform chemical, is usually produced by chemical synthesis methods. With increasingly requirement of food security and environmental protection, bio-fermentation of acetoin by microorganisms has a great promising market. However, through metabolic engineering strategies, the mixed acid-butanediol fermentation metabolizes a certain portion of substrate to the by-products of organic acids such as lactic acid and acetic acid, which causes energy cost and increases the difficulty of product purification in downstream processes. In this work, due to the high efficiency of enzymatic reaction and excellent selectivity, a strategy for efficiently converting 2,3-butandiol to acetoin using whole-cell biocatalyst by engineered Bacillus subtilis is proposed. In this process, NAD+ plays a significant role on 2,3-butanediol and acetoin distribution, so the NADH oxidase and 2,3-butanediol dehydrogenase both from B. subtilis are co-expressed in B. subtilis 168 to construct an NAD+ regeneration system, which forces dramatic decrease of the intracellular NADH concentration (1.6 fold and NADH/NAD+ ratio (2.2 fold. By optimization of the enzymatic reaction and applying repeated batch conversion, the whole-cell biocatalyst efficiently produced 91.8 g/L acetoin with a productivity of 2.30 g/(L·h, which was the highest record ever reported by biocatalysis. This work indicated that manipulation of the intracellular cofactor levels was more effective than the strategy of enhancing enzyme activity, and the bioprocess for NAD+ regeneration may also be a useful way for improving the productivity of NAD+-dependent chemistry-based products.

  19. A high-yield co-expression system for the purification of an intact Drs2p-Cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate.

    Science.gov (United States)

    Azouaoui, Hassina; Montigny, Cédric; Ash, Miriam-Rose; Fijalkowski, Frank; Jacquot, Aurore; Grønberg, Christina; López-Marqués, Rosa L; Palmgren, Michael G; Garrigos, Manuel; le Maire, Marc; Decottignies, Paulette; Gourdon, Pontus; Nissen, Poul; Champeil, Philippe; Lenoir, Guillaume

    2014-01-01

    P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼ 1-2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover a fraction that mainly contained a 1:1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated in the simultaneous presence of the transported substrate, phosphatidylserine (PS), and the regulatory lipid phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide that plays critical roles in membrane trafficking events from the trans-Golgi network (TGN). Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and

  20. Production of Functionally Active and Immunogenic Non-Glycosylated Protective Antigen from Bacillus anthracis in Nicotiana benthamiana by Co-Expression with Peptide-N-Glycosidase F (PNGase F of Flavobacterium meningosepticum.

    Directory of Open Access Journals (Sweden)

    Tarlan Mamedov

    Full Text Available Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, there is a need for a safe, low-cost and highly efficient anthrax vaccine with demonstrated long-term stability for mass vaccination in case of an emergency. Many efforts have been made towards developing an anthrax vaccine based on recombinant protective antigen (rPA of B. anthracis, a key component of the anthrax toxin, produced using different expression systems. Plants represent a promising recombinant protein production platform due to their relatively low cost, rapid scalability and favorable safety profile. Previous studies have shown that full-length rPA produced in Nicotiana benthamiana (pp-PA83 is immunogenic and can provide full protection against lethal spore challenge; however, further improvement in the potency and stability of the vaccine candidate is necessary. PA of B. anthracis is not a glycoprotein in its native host; however, this protein contains potential N-linked glycosylation sites, which can be aberrantly glycosylated during expression in eukaryotic systems including plants. This glycosylation could affect the availability of certain key epitopes either due to masking or misfolding of the protein. Therefore, a non-glycosylated form of pp-PA83 was engineered and produced in N. benthamiana using an in vivo deglycosylation approach based on co-expression of peptide-N-glycosidase F (PNGase F from Flavobacterium meningosepticum. For comparison, versions of pp-PA83 containing point mutations in six potential N-glycosylation sites were also engineered and expressed in N. benthamiana. The in vivo deglycosylated pp-PA83 (pp-dPA83 was shown to have in vitro activity, in contrast to glycosylated pp-PA83, and to induce significantly higher levels of toxin-neutralizing antibody responses in mice compared with glycosylated pp-PA83, in vitro deglycosylated pp-PA83 or the mutated versions of pp-PA83. These results suggest that pp-dPA83 may

  1. Achievement report on project in fiscal 2000 of public appeal for proposal of international joint research (new industry - No.1). Development of technology to immortalize human cells by co-expression of mortalin and telomerase; 2000 nendo kokusai kyodo kenkyu teian kobo jigyo seika hokokusho (shinki No.1). Mortalin oyobi telomerase no kyohatsugen ni yoru hitosaibo fushika gijutsu no kaihatsu

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    Researches have been performed on development of a technology to immortalize human cells by co-expression of mortalin and telomerase. The technology was adopted in the public appeal for proposal of international joint research. This paper summarizes the achievements thereof. The research has standardized a method to establish co-expression cell strains of mortalin genes and telomerase genes. Expressing the h TERT genes alone normally was not sufficient to cause immortalization in human fibroblasts. Furthermore, it was found in many cell strains that the co-expression of LTAg and h TERT is not sufficient to immortalize their cells. Expressing SV40stAg simultaneously in these cell strains was proved to have induced the immortalization highly efficiently. Introducing the mot-2 gene in place of virus antigen has resulted in acquisition of human cells regarded immortalized. It was concluded that suppressing initially the action of p53 by using mot-2 initially the immortalization due to the h TERT introduction. (NEDO)

  2. Non-structural proteins P17 and P33 are involved in the assembly of the internal membrane-containing virus PRD1

    Energy Technology Data Exchange (ETDEWEB)

    Karttunen, Jenni; Mäntynen, Sari [Centre of Excellence in Biological Interactions, Department of Biological and Environmental Science and Nanoscience Center, University of Jyväskylä, P.O. Box 35, 40014 Jyväskylä (Finland); Ihalainen, Teemu O. [Stem Cells in Neurological Applications Group, BioMediTech, University of Tampere, Tampere (Finland); Bamford, Jaana K.H. [Centre of Excellence in Biological Interactions, Department of Biological and Environmental Science and Nanoscience Center, University of Jyväskylä, P.O. Box 35, 40014 Jyväskylä (Finland); Oksanen, Hanna M., E-mail: hanna.oksanen@helsinki.fi [Institute of Biotechnology and Department of Biosciences, University of Helsinki, Biocenter 2, P.O. Box 56 (Viikinkaari 5), FIN-00014 Helsinki (Finland)

    2015-08-15

    Bacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diffusion of the P33 fluorescent fusion protein was substantially slower in E. coli than theoretically calculated, presumably resulting from intermolecular interactions. Our results indicate that P33 and P17 function in procapsid assembly, possibly in association with the host chaperonin complex GroEL/GroES. - Highlights: • Two non-structural proteins of PRD1 are involved in the virus assembly. • P17 and P33 complement the defect in GroES of Escherichia coli. • P33 co-localises with GroEL and P17 in the bacterium. • Slow motion of P33 in the bacterium suggests association with cellular components.

  3. Differential control of Salmonella heat shock operons by structured mRNAs.

    Science.gov (United States)

    Cimdins, Annika; Roßmanith, Johanna; Langklotz, Sina; Bandow, Julia E; Narberhaus, Franz

    2013-08-01

    DnaK-DnaJ-GrpE and GroES-GroEL are the major chaperone machineries in bacteria. In many species, dnaKJ and groESL are encoded in bicistronic operons. Quantitative proteomics revealed that DnaK and GroEL amounts in Salmonella dominate over DnaJ and GroES respectively. An imperfect transcriptional terminator in the intergenic region of dnaKJ is known to result in higher transcript levels of the first gene. Here, we examined the groESL operon and asked how the second gene in a heat shock operon can be preferentially expressed and found that an RNA structure in the 5'untranslated region of groES is responsible. The secondary structure masks the Shine-Dalgarno (SD) sequence and AUG start codon and thereby modulates translation of groES mRNA. Reporter gene assays combined with structure probing and toeprinting analysis revealed a dynamic temperature-sensitive RNA structure. Following an increase in temperature, only the second of two RNA hairpins melts and partially liberates the SD sequence, thus facilitating translation. Translation of groEL is not temperature-regulated leading to an excess of the chaperonin in the cell at low temperature. Discussion in a broader context shows how structured RNA segments can differentially control expression of temperature-affected operons in various ways. © 2013 John Wiley & Sons Ltd.

  4. Improvisation and co-expression in explorative digital music systems

    DEFF Research Database (Denmark)

    Hansen, Anne-Marie Skriver

    simultaneous and contrasting play forms. However, results from the quantitative analysis also show that players applied their social skills to the musical context: they were able to adapt quickly to each others’ changes in tempo and they were very flexible in terms of the distribution of musical roles. Duets...

  5. Exploring the mechanisms used by promiscuous chaperones to assist protein folding in the cell

    Science.gov (United States)

    Jewett, Andrew I.

    There are two popular theories to explain how molecular chaperones boost the yield of folded protein in the cell: According to the Anfinsen cage model, (ACM) chaperonins protect denatured proteins from aggregation. A competing theory, the iterative annealing model (IAM) claims that ATP regulated chaperone binding and release accelerates folding by freeing proteins from long-lived kinetic traps. We present experimental and kinetic evidence to argue that the IAM is not a complete picture of how the GroEL/ES chaperonin works. Surprisingly some substrate proteins experience folding rate enhancements without undergoing multiple rounds of ATP-induced binding and release from the chaperonin. An explanation of this data requires going beyond the ACM and IAM models. Our work uses molecular dynamics simulations to investigate the folding of a highly frustrated protein within a chaperonin cavity. The chaperonin interior is modeled by a sphere with variable degree of attraction to the protein inside. We demonstrate that this cavity, similar to the weakly hydrophobic interior of the GroEL cavity upon complexion with ATP and GroES, is sufficient to accelerate the folding of a frustrated protein by more than an order of magnitude. Our simulations uncover a novel form of the IAM in which the substrate exhibits spontaneous binding and release from the wall of the chaperonin cage. This mimics the behavior observed in the standard IAM, with the difference that thermal fluctuations, rather than ATP, allow the substrate to unbind from the chaperone. An growing number of smaller cageless chaperones have been discovered that can assist protein folding without the consumption of ATP, including artificial "minichaperones" (fragments of larger chaperones). It is tempting to speculate that the same thermally-driven IAM mechanism could play a role with these chaperones as well. We performed additional simulations of protein folding outside the sphere. We find that in order to accelerate

  6. Probing the functional mechanism of Escherichia coli GroEL using circular permutation.

    Directory of Open Access Journals (Sweden)

    Tomohiro Mizobata

    Full Text Available BACKGROUND: The Escherichia coli chaperonin GroEL subunit consists of three domains linked via two hinge regions, and each domain is responsible for a specific role in the functional mechanism. Here, we have used circular permutation to study the structural and functional characteristics of the GroEL subunit. METHODOLOGY/PRINCIPAL FINDINGS: Three soluble, partially active mutants with polypeptide ends relocated into various positions of the apical domain of GroEL were isolated and studied. The basic functional hallmarks of GroEL (ATPase and chaperoning activities were retained in all three mutants. Certain functional characteristics, such as basal ATPase activity and ATPase inhibition by the cochaperonin GroES, differed in the mutants while at the same time, the ability to facilitate the refolding of rhodanese was roughly equal. Stopped-flow fluorescence experiments using a fluorescent variant of the circularly permuted GroEL CP376 revealed that a specific kinetic transition that reflects movements of the apical domain was missing in this mutant. This mutant also displayed several characteristics that suggested that the apical domains were behaving in an uncoordinated fashion. CONCLUSIONS/SIGNIFICANCE: The loss of apical domain coordination and a concomitant decrease in functional ability highlights the importance of certain conformational signals that are relayed through domain interlinks in GroEL. We propose that circular permutation is a very versatile tool to probe chaperonin structure and function.

  7. Technical problems of wastewater treatment plant in crude palm oil industry A case study in PT Socfin Indonesia-Kebun Sungai Liput, Nang groe Aceh Darussalam Province

    Science.gov (United States)

    Paramitadevi, Y. V.; Rahmatullah

    2017-05-01

    Crude palm oil produced in Indonesia has already been known as the largest in the world. Unfortunately many of palm oil factories (CPOF) spread out in Indonesia have not good wastewater treatments (WWTP) yet. PT Socfin Indonesia, as an example, which is located in Aceh Tamiang Regency, still has BOD contained in its final effluent of the waswater treatment plant more than 150 ppm. In fact, the capability and capacity of WWTP in PT Socfin are 192 m3per day. Because of improper operational and maintenance of the WWTP, the technical prob lem are accumulated, such as, increasing the deposition of sludge and decreasing the retention time. The following affect is the treatment process is not going well and the quality of effluent is getting worse. The objective of this paper is to solve the technical problems by means remov ing the deposition of sludge periodically and fixing floating aeration in the aerobic pond. Method using for this paper is survey and wastewater sampling. A recommendation of the was tewater treatment system has been proposed after average BOD from WWTP outlet is defined 158 mg/L. The recommendation has seven procesess including oil separation, neutralization, closed tank anaerobic digester equipped with gas holder, extended aeration, settling tank, sand drying bed and land application.

  8. Proteomic analysis reveals metabolic and regulatory systems involved the syntrophic and axenic lifestyle of Syntrophomonas wolfei.

    Directory of Open Access Journals (Sweden)

    Jessica Rhea Sieber

    2015-02-01

    Full Text Available Microbial syntrophy is a vital metabolic interaction necessary for the complete oxidation of organic biomass to methane in all-anaerobic ecosystems. However, this process is thermodynamically constrained and represents an ecosystem-level metabolic bottleneck. To gain insight into the physiology of this process, a shotgun proteomic approach was used to quantify the protein landscape of the model syntrophic metabolizer, Syntrophomonas wolfei, grown axenically and syntrophically with Methanospirillum hungatei. Remarkably, the abundance of most proteins as represented by normalized spectral abundance factor (NSAF value changed very little between the pure and coculture growth conditions. Among the most abundant proteins detected were GroEL and GroES chaperonins, a small heat shock protein, and proteins involved in electron transfer, beta-oxidation, and ATP synthesis. Several putative energy conservation enzyme systems that utilize NADH and ferredoxin were present. The abundance of an EtfAB2 and the membrane-bound iron-sulfur oxidoreductase (Swol_0698 gene product delineated a potential conduit for electron transfer between acyl-CoA dehydrogenases and membrane redox carriers. Proteins detected only when S. wolfei was grown with M. hungatei included a zinc-dependent dehydrogenase with a GroES domain, whose gene is present in genomes in many organisms capable of syntrophy, and transcriptional regulators responsive to environmental stimuli or the physiological status of the cell. The proteomic analysis revealed an emphasis macromolecular stability and energy metabolism to S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status.

  9. Kinetic model for the coupling between allosteric transitions in GroEL and substrate protein folding and aggregation.

    Science.gov (United States)

    Tehver, Riina; Thirumalai, D

    2008-04-04

    The bacterial chaperonin GroEL and the co-chaperonin GroES assist in the folding of a number of structurally unrelated substrate proteins (SPs). In the absence of chaperonins, SP folds by the kinetic partitioning mechanism (KPM), according to which a fraction of unfolded molecules reaches the native state directly, while the remaining fraction gets trapped in a potentially aggregation-prone misfolded state. During the catalytic reaction cycle, GroEL undergoes a series of allosteric transitions (TR-->R"-->T) triggered by SP capture, ATP binding and hydrolysis, and GroES binding. We developed a general kinetic model that takes into account the coupling between the rates of the allosteric transitions and the folding and aggregation of the SP. Our model, in which the GroEL allosteric rates and SP-dependent folding and aggregation rates are independently varied without prior assumption, quantitatively fits the GroEL concentration-dependent data on the yield of native ribulose bisphosphate carboxylase/oxygenase (Rubisco) as a function of time. The extracted kinetic parameters for the GroEL reaction cycle are consistent with the available values from independent experiments. In addition, we also obtained physically reasonable parameters for the kinetic steps in the reaction cycle that are difficult to measure. If experimental values for GroEL allosteric rates are used, the time-dependent changes in native-state yield at eight GroEL concentrations can be quantitatively fit using only three SP-dependent parameters. The model predicts that the differences in the efficiencies (as measured by yields of the native state) of GroEL, single-ring mutant (SR1), and variants of SR1, in the rescue of mitochondrial malate dehydrogenase, citrate synthase, and Rubisco, are related to the large variations in the allosteric transition rates. We also show that GroEL/S mutants that efficiently fold one SP at the expense of all others are due to a decrease in the rate of a key step in the

  10. Immunoproteomics of Brucella abortus RB51 as candidate antigens in serological diagnosis of brucellosis.

    Science.gov (United States)

    Kim, Ji-Yeon; Sung, So-Ra; Lee, Kichan; Lee, Hyang-Keun; Kang, Sung-Il; Lee, Jin Ju; Jung, Suk Chan; Park, Yong Ho; Her, Moon

    2014-08-15

    The current brucellosis serodiagnostic assays are chiefly based on detecting anti-LPS (lipopolysaccharide) antibodies. However, cross-reaction with some gram-negative bacteria can occasionally induce due to similar O-polysaccharide (OPS) structure. Therefore, the aim of the present study was to identify new candidate antigens from Brucella abortus RB51, a mutant strain lacking the LPS portion, which might be valuable in brucellosis diagnosis. To detect potential antigens, immobilized pH gradients (IPG) strips with three ranges (pH 3-5.6, 4-7 and 6-11) were applied. After separating the insoluble proteins of B. abortus RB51 using two-dimensional electrophoresis (2-DE), their immunogenicity was evaluated by western blotting using four types of antisera - B. abortus, Yersinia enterocolitica O:9 and Escherichia coli O157:H7-positive, and B. abortus-negative bovine sera. Among the several immunogenic spots, the spots showing specific reactivity with only the B. abortus-positive antisera, were considered as candidate antigens. Overall, eleven immuno-reactive proteins were identified, as follows: Cu/Zn superoxide dismutase, histidinol dehydrogenase, chaperonin DnaK, chaperonin GroES, beta-ketoadipyl CoA thiolase, two-component response regulator, the cell-division protein FtsZ, aldehyde dehydrogenase, 50s ribosomal protein L10 and invasion protein B. These selected highly immunogenic protein spots might be useful as alternative antigens for brucellosis and helpful in reducing the cross-reactivity. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Physiological Responses and Gene Co-Expression Network of Mycorrhizal Roots under K+ Deprivation.

    Science.gov (United States)

    Garcia, Kevin; Chasman, Deborah; Roy, Sushmita; Ané, Jean-Michel

    2017-03-01

    Arbuscular mycorrhizal (AM) associations enhance the phosphorous and nitrogen nutrition of host plants, but little is known about their role in potassium (K+) nutrition. Medicago truncatula plants were cocultured with the AM fungus Rhizophagus irregularis under high and low K+ regimes for 6 weeks. We determined how K+ deprivation affects plant development and mineral acquisition and how these negative effects are tempered by the AM colonization. The transcriptional response of AM roots under K+ deficiency was analyzed by whole-genome RNA sequencing. K+ deprivation decreased root biomass and external K+ uptake and modulated oxidative stress gene expression in M. truncatula roots. AM colonization induced specific transcriptional responses to K+ deprivation that seem to temper these negative effects. A gene network analysis revealed putative key regulators of these responses. This study confirmed that AM associations provide some tolerance to K+ deprivation to host plants, revealed that AM symbiosis modulates the expression of specific root genes to cope with this nutrient stress, and identified putative regulators participating in these tolerance mechanisms. © 2017 American Society of Plant Biologists. All Rights Reserved.

  12. Co-expression of cyclin D1 and phosphorylated ribosomal S6 proteins in hemimegalencephaly

    NARCIS (Netherlands)

    Aronica, Eleonora; Boer, Karin; Baybis, Marianna; Yu, Jia; Crino, Peter

    2007-01-01

    Hemimegalencephaly (HMEG) is a developmental brain malformation highly associated with epilepsy. Balloon cells (BCs) and cytomegalic neurons (CNs) are frequently observed in HMEG specimens. Cytomegaly in developmental brain malformations may reflect in aberrant activation of the mTOR and

  13. Gene co-expression networks and profiles reveal potential biomarkers of boar taint in pigs

    DEFF Research Database (Denmark)

    Drag, Markus; Skinkyté-Juskiené, Rúta; Do, Duy Ngoc

    Boar taint (BT) is an offensive odour or taste of porcine meat which may occur in entire male pigs due to skatole and androstenone accumulation. To avoid BT, castration of young piglets is performed but this strategy is under debate due to animal welfare concerns. The study aimed to reveal potent...

  14. Neuropeptide co-expression in hypothalamic kisspeptin neurons of laboratory animals and the human

    Directory of Open Access Journals (Sweden)

    Katalin eSkrapits

    2015-02-01

    Full Text Available Hypothalamic peptidergic neurons using kisspeptin (KP and its co-transmitters for communication are critically involved in the regulation of mammalian reproduction and puberty. This article provides an overview of neuropeptides present in KP neurons, with a focus on the human species. Immunohistochemical studies reveal that large subsets of human KP neurons synthesize neurokinin B, as also shown in laboratory species. In contrast, dynorphin described in KP neurons of rodents and sheep is found rarely in KP cells of human males and postmenopausal females. Similarly, galanin is detectable in mouse, but not human, KP cells, whereas substance P, cocaine- and amphetamine-regulated transcript and proenkephalin-derived opioids are expressed in varying subsets of KP neurons in humans, but not reported in ARC of other species. Human KP neurons do not contain neurotensin, cholecystokinin, proopiomelanocortin-derivatives, agouti-related protein, neuropeptide Y, somatostatin or tyrosine hydroxylase (dopamine. These data identify the possible co-transmitters of human KP cells. Neurochemical properties distinct from those of laboratory species indicate that humans use considerably different neurotransmitter mechanisms to regulate fertility.

  15. Co-expression of apoptin (VP3) and antibacterial peptide cecropin B ...

    African Journals Online (AJOL)

    The antibacterial peptide cecropin B mutant (ABPS1) gene has a broad range of antibacterial and antiproliferative properties. Apoptin (VP3), a chicken anaemia virus-encoded protein is known to induce apoptosis in human transformed cells. To explore drug combination in human tumor cells, apoptin and ABPS1 eukaryotic ...

  16. Uncovering the liver’s role in immunity through RNA co-expression networks

    Czech Academy of Sciences Publication Activity Database

    Harrall, K. K.; Kechris, K. J.; Tabakoff, B.; Hoffman, P.L.; Hines, L. M.; Tsukamoto, H.; Pravenec, Michal; Printz, M.; Saba, L. M.

    2016-01-01

    Roč. 27, 9-10 (2016), s. 469-484 ISSN 0938-8990 R&D Projects: GA ČR(CZ) GAP301/12/0696 Institutional support: RVO:67985823 Keywords : RNA coexpression networks * liver * immunity * rat * recombinant inbred strains Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.509, year: 2016

  17. A co-expression gene network associated with developmental regulation of apple fruit acidity.

    Science.gov (United States)

    Bai, Yang; Dougherty, Laura; Cheng, Lailiang; Xu, Kenong

    2015-08-01

    Apple fruit acidity, which affects the fruit's overall taste and flavor to a large extent, is primarily determined by the concentration of malic acid. Previous studies demonstrated that the major QTL malic acid (Ma) on chromosome 16 is largely responsible for fruit acidity variations in apple. Recent advances suggested that a natural mutation that gives rise to a premature stop codon in one of the two aluminum-activated malate transporter (ALMT)-like genes (called Ma1) is the genetic causal element underlying Ma. However, the natural mutation does not explain the developmental changes of fruit malate levels in a given genotype. Using RNA-seq data from the fruit of 'Golden Delicious' taken at 14 developmental stages from 1 week after full-bloom (WAF01) to harvest (WAF20), we characterized their transcriptomes in groups of high (12.2 ± 1.6 mg/g fw, WAF03-WAF08), mid (7.4 ± 0.5 mg/g fw, WAF01-WAF02 and WAF10-WAF14) and low (5.4 ± 0.4 mg/g fw, WAF16-WAF20) malate concentrations. Detailed analyses showed that a set of 3,066 genes (including Ma1) were expressed not only differentially (P FDR apple fruit acidity in 'Golden Delicious'.

  18. Construction and comparison of gene co-expression networks shows complex plant immune responses

    National Research Council Canada - National Science Library

    Leal, Luis Guillermo; López, Camilo; López-Kleine, Liliana

    2014-01-01

    .... After the evaluation of diverse similarity metrics for the GCN reconstruction, we recommended the mutual information coefficient measurement and a clustering coefficient-based method for similarity threshold...

  19. Uncovering co-expression gene network regulating fruit acidity in diverse apples

    Science.gov (United States)

    Acidity is a major contributor to fruit quality. Several organic acids are present in apple fruit, but malic acid is predominant and determines fruit acidity. The trait is largely controlled by the Malic acid (Ma) locus, underpinning which Ma1 that encodes an Aluminum-activated Malate Transporter1 (...

  20. Gene co-expression network analysis identifies porcine genes associated with variation in Salmonella shedding

    OpenAIRE

    Kommadath, Arun; Bao, Hua; Arantes, Adriano S.; Plastow, Graham S; Tuggle, Christopher K.; Bearson, Shawn MD; Luo Guan, Le; Stothard, Paul

    2014-01-01

    Background Salmonella enterica serovar Typhimurium is a gram-negative bacterium that can colonise the gut of humans and several species of food producing farm animals to cause enteric or septicaemic salmonellosis. While many studies have looked into the host genetic response to Salmonella infection, relatively few have used correlation of shedding traits with gene expression patterns to identify genes whose variable expression among different individuals may be associated with differences in ...

  1. Gene co-expression network analysis for identifying modules and functionally enriched pathways in SCA2.

    Science.gov (United States)

    Pflieger, Lance T; Dansithong, Warunee; Paul, Sharan; Scoles, Daniel R; Figueroa, Karla P; Meera, Pratap; Otis, Thomas S; Facelli, Julio C; Pulst, Stefan M

    2017-08-15

    Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the ATXN2 gene. The repeat resides in an encoded region of the gene resulting in polyglutamine (polyQ) expansion which has been assumed to result in gain of function, predominantly, for the ATXN2 protein. We evaluated temporal cerebellar expression profiles by RNA sequencing of ATXN2Q127 mice versus wild-type (WT) littermates. ATXN2Q127 mice are characterized by a progressive motor phenotype onset, and have progressive cerebellar molecular and neurophysiological (Purkinje cell firing frequency) phenotypes. Our analysis revealed previously uncharacterized early and progressive abnormal patterning of cerebellar gene expression. Weighted Gene Coexpression Network Analysis revealed four gene modules that were significantly correlated with disease status, composed primarily of genes associated with GTPase signaling, calcium signaling and cell death. Of these genes, few overlapped with differentially expressed cerebellar genes that we identified in Atxn2-/- knockout mice versus WT littermates, suggesting that loss-of-function is not a significant component of disease pathology. We conclude that SCA2 is a disease characterized by gain of function for ATXN2. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Characterization of Chemically Induced Liver Injuries Using Gene Co-Expression Modules

    Science.gov (United States)

    2014-09-16

    characteristic genes for the two injury indicators, as well as functional gene annotations from the Rat Genome Database ( RGD ) [56]. In the case of...B1 (C. elegans) Antigen processing and presentation of exogenous peptide antigen via MHC class II; intracellular protein transport; toll-like receptor

  3. Integrative network biology: graph prototyping for co-expression cancer networks.

    Directory of Open Access Journals (Sweden)

    Karl G Kugler

    Full Text Available Network-based analysis has been proven useful in biologically-oriented areas, e.g., to explore the dynamics and complexity of biological networks. Investigating a set of networks allows deriving general knowledge about the underlying topological and functional properties. The integrative analysis of networks typically combines networks from different studies that investigate the same or similar research questions. In order to perform an integrative analysis it is often necessary to compare the properties of matching edges across the data set. This identification of common edges is often burdensome and computational intensive. Here, we present an approach that is different from inferring a new network based on common features. Instead, we select one network as a graph prototype, which then represents a set of comparable network objects, as it has the least average distance to all other networks in the same set. We demonstrate the usefulness of the graph prototyping approach on a set of prostate cancer networks and a set of corresponding benign networks. We further show that the distances within the cancer group and the benign group are statistically different depending on the utilized distance measure.

  4. SUMO co-expression modifies KV 11.1 channel activity

    DEFF Research Database (Denmark)

    Steffensen, Annette Buur; Andersen, Martin Nybo; Mutsaers, Nancy

    2017-01-01

    AIM: The voltage-gated potassium channel KV 11.1 is the molecular basis for the IKr current which plays an important role in cardiac physiology. Its malfunction is associated with both inherited and acquired cardiac arrhythmias. Native currents differ from those in experimental models, suggesting...... additional regulatory mechanisms. We hypothesised that the post-translational modification sumoylation finetunes channel activity. METHODS: The functional effects of sumoylation on KV 11.1 were addressed by employing two-electrode voltage-clamp (TEVC) experiments in Xenopus laevis oocytes. Site...

  5. Co-expression of the C-terminal domain of Yersinia enterocolitica ...

    Indian Academy of Sciences (India)

    2015-01-11

    Jan 11, 2015 ... infection and persistent infection), which causes great eco- nomic losses to livestock (Moennig 2000). ... targeting delivery of antigen to the lymphatic tissues (Clark et al. 1998). This characteristic is used to ... kidney cell line PK-15. The virulent CSFV. 'Shimen' strain was provided by the Control Institute of.

  6. In vivo deglycosylation of recombinant proteins in plants by co-expression with bacterial PNGase F.

    Science.gov (United States)

    Mamedov, Tarlan; Yusibov, Vidadi

    2013-01-01

    At present, several eukaryotic expression systems including yeast, insect and mammalian cells and plants are used for the production of recombinant proteins. Proteins with potential N-glycosylation sites are efficiently glycosylated when expressed in these systems. However, the ability of the eukaryotic expression systems to glycosylate may be not desirable for some proteins. If target proteins that do not carry N-linked glycans in the native host contain potential N-linked glycosylation sites, they can be aberrantly glycosylated in the eukaryotic expression systems, thus, potentially impairing biological activity. Recently, we have developed a strategy of enzymatic deglycosylation of proteins in vivo by co-introducing bacterial PNGase F via agroinfiltration followed by transient expression in plants. (1) Here, we summarize our work on this topic and its potential implications.

  7. In vivo deglycosylation of recombinant proteins in plants by co-expression with bacterial PNGase F

    OpenAIRE

    Mamedov, Tarlan; Yusibov, Vidadi

    2013-01-01

    At present, several eukaryotic expression systems including yeast, insect and mammalian cells and plants are used for the production of recombinant proteins. Proteins with potential N-glycosylation sites are efficiently glycosylated when expressed in these systems. However, the ability of the eukaryotic expression systems to glycosylate may be not desirable for some proteins. If target proteins that do not carry N-linked glycans in the native host contain potential N-linked glycosylation site...

  8. Co-expression of CD133(+)/CD44(+) in human colon cancer and liver metastasis.

    Science.gov (United States)

    Bellizzi, Antonia; Sebastian, Sinto; Ceglia, Pasquale; Centonze, Matteo; Divella, Rosa; Manzillo, Elvira Foglia; Azzariti, Amalia; Silvestris, Nicola; Montemurro, Severino; Caliandro, Cosimo; De Luca, Raffaele; Cicero, Giuseppe; Rizzo, Sergio; Russo, Antonio; Quaranta, Michele; Simone, Giovanni; Paradiso, Angelo

    2013-02-01

    Although relatively good therapeutic results are achieved in non-advanced cancer, the prognosis of the advanced colon cancer still remains poor, dependent on local or distant recurrence of the disease. One of the factors responsible for recurrence is supposed to be cancer stem cells (CSCs) or tumor-initiating cells, which are a population of cancer cells with ability to perpetuate themselves through self-renewal and to generate differentiated cells, thought to be responsible for tumor recurrence. This study globally approach the possible role of tissue-derived stem cells in the initiation of colon cancer and its metastatic process in the liver. Fresh surgical specimens from colon cancer, non-tumor tissue and liver metastasis were obtained directly from the operating room, examined, and immediately processed. CSCs were selected under serum-free conditions and characterized by CD44 and CD133 expression levels. CD133(+)/CD44(+) cell populations were then investigated in paraffin-embedded tissues and circulating tumor cells isolated from peripheral blood of the same group of colon cancer patients. Our data demonstrate that metastatic properties of cell populations from blood and liver metastasis, differently from primitive tumors, seem to be strictly related to the phenotype CD133 positive and CD44 positive. Copyright © 2012 Wiley Periodicals, Inc.

  9. Co-expression of the C-terminal domain of Yersinia enterocolitica ...

    Indian Academy of Sciences (India)

    Author Affiliations. Helin Li1 Pengbo Ning1 Zhi Lin1 Wulong Liang1 Kai Kang1 Lei He2 Yanming Zhang1. College of Veterinary Medicine, Northwest A & F University, Yangling 712100, Shaanxi, China; College of Animal Science & Technology, Henan University of Science and Technology, Luoyang 471023, Henan, China ...

  10. Chaperonin-GroEL as a Smart Hydrophobic Drug Delivery and Tumor Targeting Molecular Machine for Tumor Therapy.

    Science.gov (United States)

    Yuan, Yi; Du, Chong; Sun, Cuiji; Zhu, Jin; Wu, Shan; Zhang, Yinlong; Ji, Tianjiao; Lei, Jianlin; Yang, Yinmo; Gao, Ning; Nie, Guangjun

    2018-02-14

    The targeted delivery of hydrophobic therapeutic drugs to tumors is one of the major challenges in drug development. The use of natural proteins as drug delivery vehicles holds great promise due to various functionalities of proteins. In the current study, we exploited a natural protein, GroEL, which possesses a double layer cage structure, as a hydrophobic drug container, which is switchable by ATP binding to a hydrophilic status, to design a novel and intelligent hydrophobic drug delivery molecular machine with a controlled drug release profile. When loaded with the hydrophobic antitumor drug, Doxorubicin (Dox), GroEL was able to shield the drug from the aqueous phase of blood, releasing the drug once in the presence of a critical concentration of ATP at the tumor site. Unexpectedly, we found that GroEL has a specific affinity for the cell structural protein, plectin, which is expressed at abnormally elevated levels on the membranes of tumor cells but not in normal cells. This finding, in combination with the ATP sensitivity, makes GroEL a superior natural tumor targeting nanocarrier. Our data show that GroEL-Dox is able to effectively, and highly selectively, deliver the hydrophobic drug to fast growing tumors without overt adverse effects on the major organs. GroEL is therefore a promising drug delivery platform that can overcome the obstacles to hydrophobic drug targeting and delivery.

  11. Single-molecule detection of chaperonin dynamics through polarization rotation modulation of CdSe QD luminescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Tani, Toshiro, E-mail: ttani@cc.tuat.ac.jp [Division of Advanced Applied Physics, Tokyo University of Agriculture and Technology, Institute of Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Oda, Masaru [Division of Advanced Applied Physics, Tokyo University of Agriculture and Technology, Institute of Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Araki, Daisuke; Miyashita, Tatsuki; Nakajima, Koudai [Department of Applied Physics, Tokyo University of Agriculture and Technology, Institute of Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Arita, Mayuno [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Institute of Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Yohda, Masafumi [Division of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Institute of Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan)

    2014-08-01

    We report our recent trials examining the single-molecule three-dimensional (3D) detection of protein conformational dynamics at room temperature. Using molecular chaperones as model proteins and cadmium selenide (CdSe) semiconductor quantum dots (QDs) as nanometer-scale probes, we monitored the temporal evolution of ATP-induced conformation changes with a total internal reflection fluorescence (TIRF) microscopy imaging technique in buffer solutions. The two-dimensional (2D) degenerate nature of the emission dipoles of the QDs, due to the uniaxial wurtzite crystal structure, made it possible to capture the 3D orientation using a polarization modulation technique in real time. The temporal resolution was half the period of analyzer rotation. Although still insufficient, the obtained signals suggest possible 3D detection of specific motions, which supports the two-step conformational changes triggered by ATP attachment. - Highlights: • We report our recent trials examining the single-molecule three-dimensional (3D) detection of protein conformational dynamics at room temperature. • Using molecular chaperones as model proteins and cadmium selenide (CdSe) semiconductor quantum dots (QDs) as nanometer-scale probes, we monitored the temporal evolution of ATP-induced conformation changes with a total internal reflection fluorescence (TIRF) microscopy imaging technique in buffer solutions. • The two-dimensional (2D) degenerate nature of the emission dipoles of the QDs, due to the uniaxial wurtzite crystal structure, made it possible to capture the 3D orientation using a polarization modulation technique in real time. • The temporal resolution was half the period of analyzer rotation. • Although still insufficient, the obtained signals suggest possible 3D detection of specific motions, which supports the two-step conformational changes triggered by ATP attachment.

  12. Co-expression of periostin and EGFR in patients with esophageal squamous cell carcinoma and their prognostic significance

    Directory of Open Access Journals (Sweden)

    Jia W

    2016-08-01

    Full Text Available Wei Jia,1 Wei Wang,1 Chu-shu Ji,1 Jun-yang Niu,2 Ya-jing Lv,1 Hang-cheng Zhou,2 Bing Hu1 1Department of Medical Oncology, 2Department of Pathology, Anhui Provincial Hospital, Anhui Medical University, Hefei, People’s Republic of China Background: Both periostin (PN and epidermal growth factor receptor (EGFR can predict the prognosis of several carcinomas alone. However, coexpression of PN and EGFR in esophageal squamous cell carcinoma (ESCC still remains unknown. We aimed to clarify their relationship with clinicopathological factors and prognostic significance of their coexpression in ESCC. Patients and methods: In this single-center retrospective study, immunohistochemistry was performed to evaluate the expression of PN and EGFR in ESCC and paracarcinomatous tissues of 83 patients. The quantitative expression levels of PN and EGFR were examined in two ESCC and tumor-adjacent tissues. The levels of PN and EGFR expression were correlated with clinicopathological parameters by the χ2 or Kruskal–Wallis method. Spearman’s rank correlation test was performed to determine the relationship between PN and EGFR expression levels. Kaplan–Meier and Cox regression analyses were used to detect the prognostic factors of disease-free survival (DFS and overall survival (OS. Results: The high expression of PN protein in ESCC tissues was significantly associated with tumor length (P=0.044, differentiation grade (P=0.003, venous invasion (P=0.010, invasion depth (P=0.007, lymphatic metastasis (P=0.000, and tumor stage (P=0.000. The high expression of EGFR protein in ESCC tissues was only significantly related to lymphatic metastasis (P=0.000, invasion depth (P=0.022, and tumor stage (P=0.000. Kaplan–Meier analysis showed that high expression of PN was closely correlated to reduced OS (P=0.000 and DFS (P=0.000, which was consistent with EGFR expression. Cox regression analysis identified PN and EGFR as independent poor prognostic factors of OS and DFS in the ESCC patients (P<0.05. Moreover, the risk of death for the ESCC patients with low expression of two biomarkers and high expression of single biomarker was 0.243 times (P=0.000 and 0.503 times (P=0.030, respectively, than that for patients with high expression of two biomarkers. Conclusion: PN and EGFR are related to miscellaneous clinicopathologic characteristics. Coexpression of PN and EGFR is more closely to be of predictive value on ESCC development and progression, which may offer a novel and potential target strategy for ESCC treatment in the future. Keywords: esophageal squamous cell carcinoma, periostin, epidermal growth factor receptor, prognosis

  13. Co-expression of HER3 and MUC1 is associated with a favourable prognosis in patients with bladder cancer

    DEFF Research Database (Denmark)

    Nielsen, Trine O; Borre, Michael; Nexo, Ebba

    2015-01-01

    OBJECTIVES: To investigate the functional impact of the interaction of MUC1 with the epidermal growth factor receptors HER3 and HER4 in patients with bladder cancer. PATIENTS AND METHODS: Using reverse transcription quantitative polymerase chain reaction, we examined MUC1 expression in 82 bladder...... the prognostic value of MUC1 (P = 0.488). MUC1 expression had no correlation with survival, tumour stage or grade, or to the prognostic value of HER4. CONCLUSIONS: A high MUC1 expression was associated with a favourable prognosis in patients with bladder cancer when the expression of HER3 was also high....... This suggests an involvement of HER3 in MUC1 function in bladder cancer....

  14. brain-coX: investigating and visualising gene co-expression in seven human brain transcriptomic datasets.

    Science.gov (United States)

    Freytag, Saskia; Burgess, Rosemary; Oliver, Karen L; Bahlo, Melanie

    2017-06-08

    The pathogenesis of neurological and mental health disorders often involves multiple genes, complex interactions, as well as brain- and development-specific biological mechanisms. These characteristics make identification of disease genes for such disorders challenging, as conventional prioritisation tools are not specifically tailored to deal with the complexity of the human brain. Thus, we developed a novel web-application-brain-coX-that offers gene prioritisation with accompanying visualisations based on seven gene expression datasets in the post-mortem human brain, the largest such resource ever assembled. We tested whether our tool can correctly prioritise known genes from 37 brain-specific KEGG pathways and 17 psychiatric conditions. We achieved average sensitivity of nearly 50%, at the same time reaching a specificity of approximately 75%. We also compared brain-coX's performance to that of its main competitors, Endeavour and ToppGene, focusing on the ability to discover novel associations. Using a subset of the curated SFARI autism gene collection we show that brain-coX's prioritisations are most similar to SFARI's own curated gene classifications. brain-coX is the first prioritisation and visualisation web-tool targeted to the human brain and can be freely accessed via http://shiny.bioinf.wehi.edu.au/freytag.s/ .

  15. Microarray and gene co-expression analysis reveals that melatonin attenuates immune responses and modulates actin rearrangement in macrophages.

    Science.gov (United States)

    Kadena, Miki; Kumagai, Yutaro; Vandenbon, Alexis; Matsushima, Hitomi; Fukamachi, Haruka; Maruta, Noboru; Kataoka, Hideo; Arimoto, Takafumi; Morisaki, Hirobumi; Funatsu, Takahiro; Kuwata, Hirotaka

    2017-04-01

    Melatonin produced by the pineal gland suppresses inflammatory responses in innate immune cells. However, the mechanism of how melatonin affects inflammatory gene regulation remains unclear. Here we performed comprehensive microarray analysis combined with transcription factor binding site (TFBS) analysis using LPS-induced mouse macrophages to investigate the effect of melatonin treatment. The results showed that melatonin preferentially downregulated interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) related signaling. The results also showed that melatonin strongly suppressed virus infection related gene expression. Furthermore, TFBS analysis implicated that melatonin downregulated the binding activity of hypoxia inducible factors (HIFs), following destabilizing actin cytoskeleton which are indispensable for induction of the TRIF-dependent signaling pathway. Indeed, it was demonstrated that melatonin treatment caused impaired phagocytosis in macrophages. Thus, melatonin regulates inflammatory responses by inhibiting specific subsets of transcription factors (TFs) by disrupting actin dynamics in the macrophage. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Co-expression and prognostic value of gross cystic disease fluid protein 15 and mammaglobin in primary breast cancer.

    Science.gov (United States)

    Fritzsche, F R; Thomas, A; Winzer, K-J; Beyer, B; Dankof, A; Bellach, J; Dahl, E; Dietel, M; Kristiansen, G

    2007-11-01

    Gross cystic disease fluid protein (GCDFP-15) and mammaglobin are both widely used and accepted markers for epithelia of breast origin. We aimed to evaluate their relation of expression on parallel whole tissue sections in primary breast cancer by immunohistochemistry and also to correlate it with clinico-pathological parameters including patient survival. Primary breast carcinomas from 165 patients with a mean clinical follow-up of 73 months were immunostained using commercially available antibodies against GCDFP-15 and mammaglobin. An immunoreactive score (IRS) was calculated based on the cytoplasmic staining intensity and the number of cells stained. Cytoplasmic expression of GCDFP-15 and mammaglobin was observed in 73.3% and 72.1% of invasive breast carcinomas respectively. 91.8% of breast cancer cases expressed at least one of both markers. Both markers strongly correlated with each other and were significantly associated with lower tumour grading. Additionally, GCDFP-15 negativity was significantly associated with shortened disease-free survival times in univariate and multivariate analyses. We demonstrated the strong correlation of GCDFP-15 and mammaglobin with each other and showed that only very few primary breast cancers are completely negative for both markers. The significantly longer disease free survival times for patients with GCDFP-15 positive tumours clearly warrants further study.

  17. Induction of a common microglia gene expression signature by aging and neurodegenerative conditions : a co-expression meta-analysis

    NARCIS (Netherlands)

    Holtman, Inge R; Raj, Divya D; Miller, Jeremy A.; Schaafsma, Wandert; Yin, Zhuoran; Brouwer, Nieske; Wes, Paul D; Möller, Thomas; Orre, Marie; Kamphuis, Willem; Hol, EM|info:eu-repo/dai/nl/F-1891-2013; Boddeke, Erik W G M; Eggen, Bart J L

    2015-01-01

    INTRODUCTION: Microglia are tissue macrophages of the central nervous system that monitor brain homeostasis and react upon neuronal damage and stress. Aging and neurodegeneration induce a hypersensitive, pro-inflammatory phenotype, referred to as primed microglia. To determine the gene expression

  18. Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean

    OpenAIRE

    Guo, Bingfu; Guo, Yong; Hong, Huilong; Jin, Longguo; Zhang, Lijuan; Chang, Ru-Zhen; Lu, Wei; Lin, Min; Qiu, Li-Juan

    2015-01-01

    Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-...

  19. Co-expression of tissue factor and IL-6 in immature endothelial cells of cerebral cavernous malformations.

    Science.gov (United States)

    Noshiro, Shouhei; Mikami, Takeshi; Kataoka-Sasaki, Yuko; Sasaki, Masanori; Ohnishi, Hirofumi; Ohtaki, Shunya; Wanibuchi, Masahiko; Mikuni, Nobuhiro; Kocsis, Jeffery D; Honmou, Osamu

    2017-03-01

    Cerebral cavernous malformations (CCMs) are congenital abnormal clusters of capillaries that are prone to leaking and thought to result from a disorder of endothelial cells. The underlying pathology of CCM is not fully understood. We analyzed the expression of tissue factor (TF) and interleukin-6 (IL-6) in CCMs to determine the association of TF and IL-6 with clinical and pathological findings. Thirteen cases of operative specimens of sporadic CCMs were included in this study. The expression of messenger RNA of TF and IL-6 was assayed and the association with clinical factors was investigated. Then, the distribution of TF and IL-6 was examined with immunofluorescence. The mRNA expression of TF of CCMs was significantly higher than that of the control (p=0.017), and was correlated with the number of hemorrhage appearances (p=0.190, ρ=0.62). The mRNA expression level of IL-6 was significantly correlated with the mRNA expression level of TF (p=0.034, ρ=0.58). Examination of immunostained sections indicated that TF(+) cells were also positive for IL-6, and distributed around normal endothelial cells. Moreover, the TF(+)/IL-6(+) cells expressed CD31 and VEGFR2. The expressions of IL-6 and TF were correlated, and both were present in the same immature endothelial cells. TF is elevated in CCM and might mediate progressive events. These factors may play a prognostic role in CCM. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Induction of cotton ovule culture fibre branching by co-expression of cotton BTL, cotton SIM, and Arabidopsis STI genes.

    Science.gov (United States)

    Wang, Gaskin; Feng, Hongjie; Sun, Junling; Du, Xiongming

    2013-11-01

    The highly elongated single-celled cotton fibre consists of lint and fuzz, similar to the Arabidopsis trichome. Endoreduplication is an important determinant in Arabidopsis trichome initiation and morphogenesis. Fibre development is also controlled by functional homologues of Arabidopsis trichome patterning genes, although fibre cells do not have a branched shape like trichomes. The identification and characterization of the homologues of 10 key Arabidopsis trichome branching genes in Gossypium arboreum are reported here. Nuclear ploidy of fibres was determined, and gene function in cotton callus and fibre cells was investigated. The results revealed that the nuclear DNA content was constant in fuzz, whereas a limited and reversible change occurred in lint after initiation. Gossypeum arboreum branchless trichomes (GaBLT) was not transcribed in fibres. The homologue of STICHEL (STI), which is essential for trichome branching, was a pseudogene in Gossypium. Targeted expression of GaBLT, Arabidopsis STI, and the cytokinesis-repressing GaSIAMESE in G. hirsutum fibre cells cultured in vitro resulted in branching. The findings suggest that the distinctive developmental mechanism of cotton fibres does not depend on endoreduplication. This important component may be a relic function that can be activated in fibre cells.

  1. Co-expression of sulphydryl oxidase and protein disulphide isomerase in Escherichia coli allows for production of soluble CRM197

    CSIR Research Space (South Africa)

    Roth, Robyn L

    2017-04-01

    Full Text Available The aim of this article is to investigate the production of soluble cross-reacting material 197 (CRM(sub197)) in Escherichia coli, a safe and effective T-cell-dependent protein carrier for polysaccharides used in the manufacture and application...

  2. The Role of Eif6 in Skeletal Muscle Homeostasis Revealed by Endurance Training Co-expression Networks

    Directory of Open Access Journals (Sweden)

    Kim Clarke

    2017-11-01

    Full Text Available Regular endurance training improves muscle oxidative capacity and reduces the risk of age-related disorders. Understanding the molecular networks underlying this phenomenon is crucial. Here, by exploiting the power of computational modeling, we show that endurance training induces profound changes in gene regulatory networks linking signaling and selective control of translation to energy metabolism and tissue remodeling. We discovered that knockdown of the mTOR-independent factor Eif6, which we predicted to be a key regulator of this process, affects mitochondrial respiration efficiency, ROS production, and exercise performance. Our work demonstrates the validity of a data-driven approach to understanding muscle homeostasis.

  3. Differential expression and co-expression gene networks reveal candidate biomarkers of boar taint in non-castrated pigs

    DEFF Research Database (Denmark)

    Drag, Markus; Skinkyté-Juskiené, Ruta; Do, Duy N.

    2017-01-01

    metabolism (GSTO1, GSR, FMO3) of skatole and androstenone in liver to steroidgenesis (HSD17B7, HSD17B8, CYP27A1), regulation of steroidgenesis (STARD10, CYB5R3) and GnRH signalling (MAPK3, MAP2K2, MAP3K2) in testis. Overrepresented pathways included "Ribosome", "Protein export" and "Oxidative phosphorylation...

  4. Shared Pathways Among Autism Candidate Genes Determined by Co-expression Network Analysis of the Developing Human Brain Transcriptome

    NARCIS (Netherlands)

    Mahfouz, A.; Ziats, M.N.; Rennert, O.M.; Lelieveldt, B.P.F.; Reinders, M.J.T.

    2015-01-01

    Autism spectrum disorder (ASD) is a neurodevelopmental syndrome known to have a significant but complex genetic etiology. Hundreds of diverse genes have been implicated in ASD; yet understanding how many genes, each with disparate function, can all be linked to a single clinical phenotype remains

  5. Co-expression of tumor antigen and interleukin-2 from an adenoviral vector augments the efficiency of therapeutic tumor vaccination

    DEFF Research Database (Denmark)

    Jensen, Benjamin Anderschou Holbech; Steffensen, Maria Abildgaard; Nørgaard Nielsen, Karen

    2014-01-01

    We have previously shown that for the majority of antigens, adenoviral vaccines expressing the target antigen fused to the MHC associated invariant chain (Ii) induce an accelerated, augmented, and prolonged transgene-specific CD8+ T-cell response. Here we describe a new adenoviral vaccine vector...... tailored to meet specific demands: in the context of therapeutic vaccination, the capacity to promote an augmented effector T-cell response.Molecular Therapy (2014); doi:10.1038/mt.2014.130....

  6. Identification of putative regulatory motifs in the upstream regions of co-expressed functional groups of genes in Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Joshi NV

    2009-01-01

    Full Text Available Abstract Background Regulation of gene expression in Plasmodium falciparum (Pf remains poorly understood. While over half the genes are estimated to be regulated at the transcriptional level, few regulatory motifs and transcription regulators have been found. Results The study seeks to identify putative regulatory motifs in the upstream regions of 13 functional groups of genes expressed in the intraerythrocytic developmental cycle of Pf. Three motif-discovery programs were used for the purpose, and motifs were searched for only on the gene coding strand. Four motifs – the 'G-rich', the 'C-rich', the 'TGTG' and the 'CACA' motifs – were identified, and zero to all four of these occur in the 13 sets of upstream regions. The 'CACA motif' was absent in functional groups expressed during the ring to early trophozoite transition. For functional groups expressed in each transition, the motifs tended to be similar. Upstream motifs in some functional groups showed 'positional conservation' by occurring at similar positions relative to the translational start site (TLS; this increases their significance as regulatory motifs. In the ribonucleotide synthesis, mitochondrial, proteasome and organellar translation machinery genes, G-rich, C-rich, CACA and TGTG motifs, respectively, occur with striking positional conservation. In the organellar translation machinery group, G-rich motifs occur close to the TLS. The same motifs were sometimes identified for multiple functional groups; differences in location and abundance of the motifs appear to ensure different modes of action. Conclusion The identification of positionally conserved over-represented upstream motifs throws light on putative regulatory elements for transcription in Pf.

  7. Systems Toxicology of Chemically Induced Liver and Kidney Injuries: Histopathology-Associated Gene Co-Expression Modules

    Science.gov (United States)

    2016-01-04

    2016 (wileyonlinelibrary.com) DOI 10.1002/jat.3278Systems toxicology of chemically induced liver and kidney injuries: histopathology -associated gene...Mapping chemical injuries to organ-specific histopathology outcomes via biomarkers will provide a foundation for designing precise and robust diagnostic...exposures and adverse histopathology assessments in Sprague–Dawley rats. We proposed a protocol for selecting gene modules associated with chemical-induced

  8. B-Lymphoblastic Lymphomas Evolving from Follicular Lymphomas Co-Express Surrogate Light Chains and Mutated Gamma Heavy Chains

    NARCIS (Netherlands)

    Slot, L.M.; Hoogeboom, R.; Smit, L.A.; Wormhoudt, T.A.M.; Biemond, B.J.; Oud, M.E.C.M.; Schilder-Tol, E.J.M.; Mulder, A.B.; Jongejan, A.; van Kampen, A.H.C.; Kluin, P.M.; Guikema, J.E.J.; Bende, R.J.; van Noesel, C.J.M.

    2016-01-01

    Follicular lymphoma (FL) is an indolent B-cell non-Hodgkin lymphoma able to transform into germinal center-type diffuse large B-cell lymphoma. We describe four extraordinary cases of FL, which progressed to TdT+CD20- precursor B-lymphoblastic lymphoma (B-LBL). Fluorescence in situ hybridization

  9. Immunocytochemical evidence for co-expression of Type III IP3 receptor with signaling components of bitter taste transduction

    Directory of Open Access Journals (Sweden)

    Kinnamon Sue C

    2001-04-01

    Full Text Available Abstract Background Taste receptor cells are responsible for transducing chemical stimuli into electrical signals that lead to the sense of taste. An important second messenger in taste transduction is IP3, which is involved in both bitter and sweet transduction pathways. Several components of the bitter transduction pathway have been identified, including the T2R/TRB taste receptors, phospholipase C β2, and the G protein subunits α-gustducin, β3, and γ13. However, the identity of the IP3 receptor subtype in this pathway is not known. In the present study we used immunocytochemistry on rodent taste tissue to identify the IP3 receptors expressed in taste cells and to examine taste bud expression patterns for IP3R3. Results Antibodies against Type I, II, and III IP3 receptors were tested on sections of rat and mouse circumvallate papillae. Robust cytoplasmic labeling for the Type III IP3 receptor (IP3R3 was found in a large subset of taste cells in both species. In contrast, little or no immunoreactivity was seen with antibodies against the Type I or Type II IP3 receptors. To investigate the potential role of IP3R3 in bitter taste transduction, we used double-label immunocytochemistry to determine whether IP3R3 is expressed in the same subset of cells expressing other bitter signaling components. IP3R3 immunoreactive taste cells were also immunoreactive for PLCβ2 and γ13. Alpha-gustducin immunoreactivity was present in a subset of IP3R3, PLCβ2, and γ13 positive cells. Conclusions IP3R3 is the dominant form of the IP3 receptor expressed in taste cells and our data suggest it plays an important role in bitter taste transduction.

  10. Model-Based Analysis of HER Activation in Cells Co-Expressing EGFR, HER2 and HER3

    OpenAIRE

    Harish Shankaran; Yi Zhang; Yunbing Tan; Haluk Resat

    2013-01-01

    The HER/ErbB family of receptor tyrosine kinases drives critical responses in normal physiology and cancer, and the expression levels of the various HER receptors are critical determinants of clinical outcomes. HER activation is driven by the formation of various dimer complexes between members of this receptor family. The HER dimer types can have differential effects on downstream signaling and phenotypic outcomes. We constructed an integrated mathematical model of HER activation, and traffi...

  11. Response of substances co-expressed in hypothalamic magnocellular neurons to osmotic challenges in normal and Brattleboro rats

    DEFF Research Database (Denmark)

    Bundzikova, J.; Pirnik, Z.; Zelena, D.

    2008-01-01

    The intention of this review is to emphasize the current knowledge about the extent and importance of the substances co-localized with magnocellular arginine vasopressin (AVP) and oxytocin (OXY) as potential candidates for the gradual clarification of their actual role in the regulation of hydrom......The intention of this review is to emphasize the current knowledge about the extent and importance of the substances co-localized with magnocellular arginine vasopressin (AVP) and oxytocin (OXY) as potential candidates for the gradual clarification of their actual role in the regulation...

  12. Induction of cotton ovule culture fibre branching by co-expression of cotton BTL, cotton SIM, and Arabidopsis STI genes

    OpenAIRE

    Wang, Gaskin; Feng, Hongjie; Sun, Junling; Du, Xiongming

    2013-01-01

    The highly elongated single-celled cotton fibre consists of lint and fuzz, similar to the Arabidopsis trichome. Endoreduplication is an important determinant in Arabidopsis trichome initiation and morphogenesis. Fibre development is also controlled by functional homologues of Arabidopsis trichome patterning genes, although fibre cells do not have a branched shape like trichomes. The identification and characterization of the homologues of 10 key Arabidopsis trichome branching genes in Gossypi...

  13. Engineering carbon fixation in E. coli: from heterologous RuBisCO expression to the Calvin-Benson-Bassham cycle.

    Science.gov (United States)

    Antonovsky, Niv; Gleizer, Shmuel; Milo, Ron

    2017-10-01

    Carbon fixation is the gateway of inorganic carbon into the biosphere. Our ability to engineer carbon fixation pathways in living organisms is expected to play a crucial role in the quest towards agricultural and energetic sustainability. Recent successes to introduce non-native carbon fixation pathways into heterotrophic hosts offer novel platforms for manipulating these pathways in genetically malleable organisms. Here, we focus on past efforts and future directions for engineering the dominant carbon fixation pathway in the biosphere, the Calvin-Benson cycle, into the well-known model organism Escherichia coli. We describe how central carbon metabolism of this heterotrophic bacterium can be manipulated to allow directed evolution of carbon fixing enzymes. Finally, we highlight future directions towards synthetic autotrophy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Model-based Analysis of HER Activation in Cells Co-Expressing EGFR, HER2 and HER3.

    Energy Technology Data Exchange (ETDEWEB)

    Shankaran, Harish; Zhang, Yi; Tan, Yunbing; Resat, Haluk

    2013-08-22

    The HER/ErbB family of receptor tyrosine kinases drive critical responses in normal physiology and cancer, and the expression levels of the various HER receptors are critical determinants of clinical outcomes. HER activation is driven by the formation of various dimer complexes between members of this receptor family. The HER dimer types can have differential effects on downstream signaling and phenotypic outcomes. We constructed an integrated mathematical model of HER activation and trafficking to quantitatively link receptor expression levels to dimerization and activation. We parameterized the model with a comprehensive set of HER phosphorylation and abundance data collected in a panel of human mammary epithelial cells expressing varying levels of EGFR, HER2 and HER3. Although parameter estimation yielded multiple solutions, predictions for dimer phosphorylation were in agreement with each other. We validated the model using experiments where pertuzumab was used to block HER2 dimerization. We used the model to predict HER dimerization and activation patterns in a panel of epithelial cells lines with known HER expression levels. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1/1 and HER1/2 dimers, and not HER1/3 dimers, ii) HER1/2 and HER2/3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2/3 dimer is largely responsible for HER3 activation. The model can be used to predict phosphorylated dimer levels for any given HER expression profile. This information in turn can be used to quantify the potencies of the various HER dimers, and can potentially inform personalized therapeutic approaches.

  15. ORF Alignment: NC_005140 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available VKKSNRGKVIAAGLGKRLENGERASMEVK 60 ... MNIRPLNDKLIVERQEVENKSEGGIVLTSQSVKKSNRGKVIAAGLGKRLENGERASMEVK Sbjct: 1 ... MNIRPLNDKLIVERQEVENKSEGGIVLTSQSVKKSNRGKVIAAGLGKRLENGERASMEVK 60 ... ...onin 2 (Protein Cpn10 2) (groES protein 2) ... Length = 96 ... Query: 1 ... MNIRPLNDKLIVERQEVENKSEGGIVLTSQS

  16. ORF Alignment: NC_004460 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available VKKSNRGKVIAAGLGKRLENGERASMEVK 60 ... MNIRPLNDKLIVERQEVENKSEGGIVLTSQSVKKSNRGKVIAAGLGKRLENGERASMEVK Sbjct: 1 ... MNIRPLNDKLIVERQEVENKSEGGIVLTSQSVKKSNRGKVIAAGLGKRLENGERASMEVK 60 ... ...onin 2 (Protein Cpn10 2) (groES protein 2) ... Length = 96 ... Query: 1 ... MNIRPLNDKLIVERQEVENKSEGGIVLTSQS

  17. Specificity and selectivity of HypC chaperonins and endopeptidases in the molecular assembly machinery of [NiFe] hydrogenases of Thiocapsa roseopersicina

    Energy Technology Data Exchange (ETDEWEB)

    Maroti, Gergely [BayGen Institute, Bay Zoltan Foundation for Applied Research, Derkovics fasor 2, Szeged 6726 (Hungary); Rakhely, Gabor; Kovacs, Kornel L. [Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Temesvari krt. 62., Szeged 6726 (Hungary); Department of Biotechnology, University of Szeged, Koezep fasor 52., Szeged 6726 (Hungary); Maroti, Judit [Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Temesvari krt. 62., Szeged 6726 (Hungary); Doroghazi, Emma [Department of Biotechnology, University of Szeged, Koezep fasor 52., Szeged 6726 (Hungary); Klement, Eva; Medzihradszky, Katalin F. [Proteomics Research Group, Biological Research Centre, Hungarian Academy of Sciences, Temesvari krt. 62., Szeged 6726 (Hungary)

    2010-04-15

    The purple photosynthetic bacterium, Thiocapsa roseopersicina harbours at least three functional [NiFe] hydrogenases. Two of them are attached to the periplasmic membrane (HynSL, HupSL), while the third one is apparently localized in the cytoplasm (HoxEFUYH). Two hypC-type genes, coding for putative small maturation proteins, were found and their roles were studied by activity measurements performed with hypC mutants. Protein-protein interaction experiments confirmed that each HypC-type protein participates in the maturation of at least two [NiFe] hydrogenase large subunits via direct interaction. Endopeptidases perform the last step of the complex [NiFe] hydrogenase maturation process. A separate endopeptidase (HynD, HupD, HoxW) cleaves off the C-terminus of each large subunit and they are strictly specific for their corresponding hydrogenases. The results demonstrate a sophisticated assembly of these functionally active redox metalloenzymes through specific and selective protein-protein interactions and imply some diversity in the hydrogenase assembly machinery among the various microbes. (author)

  18. Two members of the TRiC chaperonin complex, CCT2 and TCP1 are essential for survival of breast cancer cells and are linked to driving oncogenes.

    Science.gov (United States)

    Guest, Stephen T; Kratche, Zachary R; Bollig-Fischer, Aliccia; Haddad, Ramsi; Ethier, Stephen P

    2015-03-15

    Gene amplification is a common mechanism of oncogene activation in cancer. Several large-scale efforts aimed at identifying the comprehensive set of genomic regions that are recurrently amplified in cancer have been completed. In breast cancer, these studies have identified recurrently amplified regions containing known drivers such as HER2 and CCND1 as well as regions where the driver oncogene is unknown. In this study, we integrated RNAi-based functional genetic data with copy number and expression data to identify genes that are recurrently amplified, overexpressed and also necessary for the growth/survival of breast cancer cells. Further analysis using clinical data from The Cancer Genome Atlas specifically identified candidate genes that play a role in determining patient outcomes. Using this approach, we identified two genes, TCP1 and CCT2, as being recurrently altered in breast cancer, necessary for growth/survival of breast cancer cells in vitro, and determinants of overall survival in breast cancer patients. We also show that expression of TCP1 is regulated by driver oncogene activation of PI3K signaling in breast cancer. Interestingly, the TCP1 and CCT2 genes both encode for components of a multi-protein chaperone complex in the cell known as the TCP1 Containing Ring Complex (TRiC). Our results demonstrate a role for the TRiC subunits TCP1 and CCT2, and potentially the entire TRiC complex, in breast cancer and provide rationale for TRiC as a novel therapeutic target in breast cancer. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Yeast Interacting Proteins Database: YOR020C, YOR020C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YOR020C HSP10 Mitochondrial matrix co-chaperonin that inhibits the ATPase activity of Hsp60p, a mitochondrial...this bait as prey (3) YOR020C HSP10 Mitochondrial matrix co-chaperonin that inhibits the ATPase activity...gene name HSP10 Bait description Mitochondrial matrix co-chaperonin that inhibits the ATPase activity...gene name HSP10 Prey description Mitochondrial matrix co-chaperonin that inhibits the ATPase activity

  20. ORF Alignment: NC_002677 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available l Structure Of The Immunodominant ... Heat-Shock Protein Chaperonin-10 Of Mycobacterium Leprae ... ...peronin-10 Of ... Mycobacterium Leprae pdb|1LEP|E Chain E, ... Thre...e-Dimensional Structure Of The Immunodominant ... Heat-Shock Protein Chaperonin-10 Of Mycobacterium Lepra... Protein Chaperonin-10 Of ... Mycobacterium Leprae pdb|1LEP|C Chain C, ... ... ... Three-Dimensional Structure Of The Immunodominant ... Heat-Shock Protein Chaperonin-10 Of Mycobacterium Lepra

  1. Compensatory changes in GroEL/Gp31 affinity as a mechanism for allele-specific genetic interaction

    NARCIS (Netherlands)

    Richardson, A.; van der Vies, S.; Keppel, F.; Taher, A.; Landry, S.J.; Georgopoulos, C.

    1999-01-01

    Previous work has shown that the GroEL-GroES interaction is primarily mediated by the GroES mobile loop. In bacteriophage T4 infection, GroES is substituted by the gene 31-encoded cochaperonin, Gp31. Using a genetic selection scheme, we have identified a new set of mutations in gene 31 that affect

  2. NCBI nr-aa BLAST: CBRC-PMAR-01-0259 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PMAR-01-0259 ref|YP_871899.1| Alcohol dehydrogenase GroES domain protein [Acidothermus... cellulolyticus 11B] gb|ABK51913.1| Alcohol dehydrogenase GroES domain protein [Acidothermus cellulolyticus 11B] YP_871899.1 8.8 30% ...

  3. NCBI nr-aa BLAST: CBRC-PTRO-10-0009 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PTRO-10-0009 ref|YP_981446.1| Alcohol dehydrogenase GroES domain protein [Polar...omonas naphthalenivorans CJ2] gb|ABM36525.1| Alcohol dehydrogenase GroES domain protein [Polaromonas naphthalenivorans CJ2] YP_981446.1 4.6 33% ...

  4. KiSS-1 and GPR54 genes are co-expressed in rat gonadotrophs and differentially regulated in vivo by oestradiol and gonadotrophin-releasing hormone.

    Science.gov (United States)

    Richard, N; Galmiche, G; Corvaisier, S; Caraty, A; Kottler, M-L

    2008-03-01

    Kisspeptin, the product derived from KiSS-1, and its cognate receptor, GPR54, both exert a role in the neuroendocrine control of reproduction by regulating gonadotrophin-releasing hormone (GnRH) secretion. In the present study, we demonstrate, using dual immunofluorescence with specific antibodies, that the KiSS-1 and GPR54 genes are both expressed in rat gonadotrophs. All luteinising hormone beta-immunoreactive (LH beta-ir) cells were stained by the KiSS-1 antibody but some kisspeptin-ir cells were not LH beta positive; thus, we cannot exclude the possibility that kisspeptins are expressed in other pituitary cells. All GPR54-ir are co-localised with LH beta cells, but only a subset of LH beta cells are stained with the GPR54 antibody. Using the real-time reverse transcription-polymerase chain reaction (RT-PCR), we found that the expression of KiSS-1 and GPR54 is differentially regulated by steroids. In the female, KiSS-1 mRNA levels dramatically decreased following ovariectomy (OVX), and this decrease was prevented by administration of 17beta-oestradiol (E(2)), but not by administration of GnRH antagonist or agonist. Administration of E(2) in OVX rats receiving either GnRH antagonist or agonist clearly shows that E(2) acts directly on the pituitary to positively control KiSS-1 expression. In OVX rats, administration of the selective oestrogen receptor (ER)alpha ligand propylpyrazoletriol, but not the selective ER beta ligand diarylpropionitrile, mimics this effect. By contrast, our study shows that GPR54 expression is positively regulated by GnRH and negatively controlled by chronic exposure to E(2). In summary, our data document for the first time that, in the female rat pituitary, KiSS-1 expression is up-regulated by oestradiol, similarly to that seen in the anteroventral periventricular nucleus of the hypothalamus. Conversely, GPR54 is up-regulated by GnRH, which exclusively targets gonadotrophs.

  5. Bicistronic lentiviruses containing a viral 2A cleavage sequence reliably co-express two proteins and restore vision to an animal model of LCA1.

    Directory of Open Access Journals (Sweden)

    Jonathan D Verrier

    Full Text Available The disease processes underlying inherited retinal disease are complex and are not completely understood. Many of the corrective gene therapies designed to treat diseases linked to mutations in genes specifically expressed in photoreceptor cells restore function to these cells but fail to stop progression of the disease. There is growing consensus that effective treatments for these diseases will require delivery of multiple therapeutic proteins that will be selected to treat specific aspects of the disease process. The purpose of this study was to design a lentiviral transgene that reliably expresses all of the proteins it encodes and does so in a consistent manner among infected cells. We show, using both in vitro and in vivo analyses, that bicistronic lentiviral transgenes encoding two fluorescent proteins fused to a viral 2A-like cleavage peptide meet these expression criteria. To determine if this transgene design is suitable for therapeutic applications, we replaced one of the fluorescent protein genes with the gene encoding guanylate cyclase-1 (GC1 and delivered lentivirus carrying this transgene to the retinas of the GUCY1*B avian model of Leber congenital amaurosis-1 (LCA1. GUCY1*B chickens carry a null mutation in the GC1 gene that disrupts photoreceptor function and causes blindness at hatching, a phenotype that closely matches that observed in humans with LCA1. We found that treatment of these animals with the 2A lentivector encoding GC1 restored vision to these animals as evidenced by the presence of optokinetic reflexes. We conclude that 2A-like peptides, with proper optimization, can be successfully incorporated into therapeutic vectors designed to deliver multiple proteins to neural retinal. These results highlight the potential of this vector design to serve as a platform for the development of combination therapies designed to enhance or prolong the benefits of corrective gene therapies.

  6. Bicistronic lentiviruses containing a viral 2A cleavage sequence reliably co-express two proteins and restore vision to an animal model of LCA1.

    Science.gov (United States)

    Verrier, Jonathan D; Madorsky, Irina; Coggin, William E; Geesey, Mero; Hochman, Michael; Walling, Elleanor; Daroszewski, Daniel; Eccles, Kristofer S; Ludlow, Rachel; Semple-Rowland, Susan L

    2011-01-01

    The disease processes underlying inherited retinal disease are complex and are not completely understood. Many of the corrective gene therapies designed to treat diseases linked to mutations in genes specifically expressed in photoreceptor cells restore function to these cells but fail to stop progression of the disease. There is growing consensus that effective treatments for these diseases will require delivery of multiple therapeutic proteins that will be selected to treat specific aspects of the disease process. The purpose of this study was to design a lentiviral transgene that reliably expresses all of the proteins it encodes and does so in a consistent manner among infected cells. We show, using both in vitro and in vivo analyses, that bicistronic lentiviral transgenes encoding two fluorescent proteins fused to a viral 2A-like cleavage peptide meet these expression criteria. To determine if this transgene design is suitable for therapeutic applications, we replaced one of the fluorescent protein genes with the gene encoding guanylate cyclase-1 (GC1) and delivered lentivirus carrying this transgene to the retinas of the GUCY1*B avian model of Leber congenital amaurosis-1 (LCA1). GUCY1*B chickens carry a null mutation in the GC1 gene that disrupts photoreceptor function and causes blindness at hatching, a phenotype that closely matches that observed in humans with LCA1. We found that treatment of these animals with the 2A lentivector encoding GC1 restored vision to these animals as evidenced by the presence of optokinetic reflexes. We conclude that 2A-like peptides, with proper optimization, can be successfully incorporated into therapeutic vectors designed to deliver multiple proteins to neural retinal. These results highlight the potential of this vector design to serve as a platform for the development of combination therapies designed to enhance or prolong the benefits of corrective gene therapies.

  7. Augmentation of alphavirus vector-induced human papilloma virus-specific immune and anti-tumour responses by co-expression of interleukin-12

    NARCIS (Netherlands)

    Riezebos-Brilman, Annelies; Regts, Joke; Chen, Margaret; Wilschut, Jan; Daemen, Toos

    2009-01-01

    To enhance the efficacy of a therapeutic immunisition strategy against human papillomavirus-induced cervical cancer we evaluated the adjuvant effect of interleukin-12 (IL12) expressed by a Semliki Forest virus vector (SFV) in mice. Depending on the dose and schedule. SFV-IL12 Stimulated

  8. Induction of glutamine synthetase and transient co-expression with carbamoylphosphate synthetase in hepatocytes transplanted into fat pads of syngeneic hosts

    NARCIS (Netherlands)

    Gebhardt, R.; Jirtle, R.; Moorman, A. F.; Lamers, W. H.; Michalopoulos, G.

    1989-01-01

    Isolated rat hepatocytes were transplanted into the interscapular and both anterior lateral fat pads of hepatectomized syngeneic rats. At various time points following transplantation, the fat pads were removed, fixed and embedded in paraffin. Serial sections were stained for glutamine synthetase

  9. Co-expression of t(15;17) and t(8;21) in a Case of Acute Promyelocytic Leukemia: Review of the Literature

    Science.gov (United States)

    UZ, Burak; Eliaçık, Eylem; Işık, Ayse; Aksu, Salih; Büyükaşık, Yahya; Haznedaroğlu, İbrahim C.; Göker, Hakan; Sayınalp, Nilgün; Özcebe, Osman İ.

    2013-01-01

    Additional chromosomal abnormalities in acute myelogenous leukemia have been identified as one of the most important prognostic factors. Favorable chromosomal changes such as t(8;21), inv(16), and t(15;17) are associated with higher rates of complete remission and event-free survival. Translocation (15;17) characterizes acute promyelocytic leukemia (APL) (French-American-British class M3) in almost all patients. Secondary chromosomal abnormalities are also present in approximately 23%-29% of patients with newly diagnosed APL. The prognostic implications of t(8;21) and other secondary cytogenetic aberrations in APL are reviewed here. We present a 47-year-old woman diagnosed with APL whose initial cytogenetic analysis included both t(8;21) and t(15;17). The initial induction chemotherapy included 3 days of idarubicin (12 mg/m2/day) and daily all-trans retinoic acid (ATRA; 45 mg/m2/day). At the sixth week of treatment, a control bone marrow biopsy was found to be normocellular, t(15;17) bcr3 and t(8;21) were negative, and t(15;17) bcr1 fusion transcripts were reduced from 5007 (1.78525699%) copies per 1 µg RNA to 40 (0.00062020%) with real-time quantitative polymerase chain reaction. Consolidation with 4 days of idarubicin (5 mg/m2/day), ATRA (45 mg/m2/day for 15 days), and cytarabine (1 g/m2/day for 4 days) was then started. However, the patient became pancytopenic and had neutropenic fever after consolidation treatment. Unfortunately, she died 3 months after the time of APL diagnosis, due to acute respiratory distress syndrome-like respiratory problems and multiorgan dysfunction requiring respiratory support and hemodialysis. Conflict of interest:None declared. PMID:24385831

  10. High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs

    DEFF Research Database (Denmark)

    Duda, Katarzyna; Lonowski, Lindsey A; Kofoed-Nielsen, Michael

    2014-01-01

    Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing...... were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant...... nickase pairs, which exhibit low off-target mutagenesis compared to wild-type Cas9....

  11. Normalized lmQCM: An Algorithm for Detecting Weak Quasi-Cliques in Weighted Graph with Applications in Gene Co-Expression Module Discovery in Cancers.

    Science.gov (United States)

    Zhang, Jie; Huang, Kun

    2014-01-01

    In this paper, we present a new approach for mining weighted networks to identify densely connected modules such as quasi-cliques. Quasi-cliques are densely connected subnetworks in a network. Detecting quasi-cliques is an important topic in data mining, with applications such as social network study and biomedicine. Our approach has two major improvements upon previous work. The first is the use of local maximum edges to initialize the search in order to avoid excessive overlaps among the modules, thereby greatly reducing the computing time. The second is the inclusion of a weight normalization procedure to enable discovery of "subtle" modules with more balanced sizes. We carried out careful tests on multiple parameters and settings using two large cancer datasets. This approach allowed us to identify a large number of gene modules enriched in both biological functions and chromosomal bands in cancer data, suggesting potential roles of copy number variations (CNVs) involved in the cancer development. We then tested the genes in selected modules with enriched chromosomal bands using The Cancer Genome Atlas data, and the results strongly support our hypothesis that the coexpression in these modules are associated with CNVs. While gene coexpression network analyses have been widely adopted in disease studies, most of them focus on the functional relationships of coexpressed genes. The relationship between coexpression gene modules and CNVs are much less investigated despite the potential advantage that we can infer from such relationship without genotyping data. Our new approach thus provides a means to carry out deep mining of the gene coexpression network to obtain both functional and genetic information from the expression data.

  12. Green tissue-specific co-expression of chitinase and oxalate oxidase 4 genes in rice for enhanced resistance against sheath blight.

    Science.gov (United States)

    Karmakar, Subhasis; Molla, Kutubuddin Ali; Chanda, Palas K; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

    2016-01-01

    Green tissue-specific simultaneous overexpression of two defense-related genes ( OsCHI11 & OsOXO4 ) in rice leads to significant resistance against sheath blight pathogen ( R. solani ) without distressing any agronomically important traits. Overexpressing two defense-related genes (OsOXO4 and OsCHI11) cloned from rice is effective at enhancing resistance against sheath blight caused by Rhizoctonia solani. These genes were expressed under the control of two different green tissue-specific promoters, viz. maize phosphoenolpyruvate carboxylase gene promoter, PEPC, and rice cis-acting 544-bp DNA element, immediately upstream of the D54O translational start site, P D54O-544 . Putative T0 transgenic rice plants were screened by PCR and integration of genes was confirmed by Southern hybridization of progeny (T1) rice plants. Successful expression of OsOXO4 and OsCHI11 in all tested plants was confirmed. Expression of PR genes increased significantly following pathogen infection in overexpressing transgenic plants. Following infection, transgenic plants exhibited elevated hydrogen peroxide levels, significant changes in activity of ROS scavenging enzymes and reduced membrane damage when compared to their wild-type counterpart. In a Rhizoctonia solani toxin assay, a detached leaf inoculation test and an in vivo plant bioassay, transgenic plants showed a significant reduction in disease symptoms in comparison to non-transgenic control plants. This is the first report of overexpression of two different PR genes driven by two green tissue-specific promoters providing enhanced sheath blight resistance in transgenic rice.

  13. Co-expression of bacterial aspartate kinase and adenylylsulfate reductase genes substantially increases sulfur amino acid levels in transgenic alfalfa (Medicago sativa L..

    Directory of Open Access Journals (Sweden)

    Zongyong Tong

    Full Text Available Alfalfa (Medicago sativa L. is one of the most important forage crops used to feed livestock, such as cattle and sheep, and the sulfur amino acid (SAA content of alfalfa is used as an index of its nutritional value. Aspartate kinase (AK catalyzes the phosphorylation of aspartate to Asp-phosphate, the first step in the aspartate family biosynthesis pathway, and adenylylsulfate reductase (APR catalyzes the conversion of activated sulfate to sulfite, providing reduced sulfur for the synthesis of cysteine, methionine, and other essential metabolites and secondary compounds. To reduce the feedback inhibition of other metabolites, we cloned bacterial AK and APR genes, modified AK, and introduced them into alfalfa. Compared to the wild-type alfalfa, the content of cysteine increased by 30% and that of methionine increased substantially by 60%. In addition, a substantial increase in the abundance of essential amino acids (EAAs, such as aspartate and lysine, was found. The results also indicated a close connection between amino acid metabolism and the tricarboxylic acid (TCA cycle. The total amino acid content and the forage biomass tested showed no significant changes in the transgenic plants. This approach provides a new method for increasing SAAs and allows for the development of new genetically modified crops with enhanced nutritional value.

  14. Functional enhancement of AT1R potency in the presence of the TPαR is revealed by a comprehensive 7TM receptor co-expression screen

    DEFF Research Database (Denmark)

    Hansen, Jonas Tind; Lyngsø, Christina; Speerschneider, Tobias

    2013-01-01

    Functional cross-talk between seven transmembrane (7TM) receptors can dramatically alter their pharmacological properties, both in vitro and in vivo. This represents an opportunity for the development of novel therapeutics that potentially target more specific biological effects while causing few...

  15. Co-expression of foreign proteins tethered to HIV-1 envelope glycoprotein on the cell surface by introducing an intervening second membrane-spanning domain.

    Directory of Open Access Journals (Sweden)

    Hongyun Wang

    Full Text Available The envelope glycoprotein (Env of human immunodeficiency virus type I (HIV-1 mediates membrane fusion. To analyze the mechanism of HIV-1 Env-mediated membrane fusion, it is desirable to determine the expression level of Env on the cell surface. However, the quantification of Env by immunological staining is often hampered by the diversity of HIV-1 Env and limited availability of universal antibodies that recognize different Envs with equal efficiency. To overcome this problem, here we linked a tag protein called HaloTag at the C-terminus of HIV-1 Env. To relocate HaloTag to the cell surface, we introduced a second membrane-spanning domain (MSD between Env and HaloTag. The MSD of transmembrane protease serine 11D, a type II transmembrane protein, successfully relocated HaloTag to the cell surface. The surface level of Env can be estimated indirectly by staining HaloTag with a specific membrane-impermeable fluorescent ligand. This tagging did not compromise the fusogenicity of Env drastically. Furthermore, fusogenicity of Env was preserved even after the labeling with the ligands. We have also found that an additional foreign peptide or protein such as C34 or neutralizing single-chain variable fragment (scFv can be linked to the C-terminus of the HaloTag protein. Using these constructs, we were able to determine the required length of C34 and critical residues of neutralizing scFv for blocking membrane fusion, respectively.

  16. Co-expression of foreign proteins tethered to HIV-1 envelope glycoprotein on the cell surface by introducing an intervening second membrane-spanning domain.

    Science.gov (United States)

    Wang, Hongyun; Li, Xiao; Nakane, Shuhei; Liu, Shujun; Ishikawa, Hirohito; Iwamoto, Aikichi; Matsuda, Zene

    2014-01-01

    The envelope glycoprotein (Env) of human immunodeficiency virus type I (HIV-1) mediates membrane fusion. To analyze the mechanism of HIV-1 Env-mediated membrane fusion, it is desirable to determine the expression level of Env on the cell surface. However, the quantification of Env by immunological staining is often hampered by the diversity of HIV-1 Env and limited availability of universal antibodies that recognize different Envs with equal efficiency. To overcome this problem, here we linked a tag protein called HaloTag at the C-terminus of HIV-1 Env. To relocate HaloTag to the cell surface, we introduced a second membrane-spanning domain (MSD) between Env and HaloTag. The MSD of transmembrane protease serine 11D, a type II transmembrane protein, successfully relocated HaloTag to the cell surface. The surface level of Env can be estimated indirectly by staining HaloTag with a specific membrane-impermeable fluorescent ligand. This tagging did not compromise the fusogenicity of Env drastically. Furthermore, fusogenicity of Env was preserved even after the labeling with the ligands. We have also found that an additional foreign peptide or protein such as C34 or neutralizing single-chain variable fragment (scFv) can be linked to the C-terminus of the HaloTag protein. Using these constructs, we were able to determine the required length of C34 and critical residues of neutralizing scFv for blocking membrane fusion, respectively.

  17. Investigating the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system / Johannes Frederik Wentzel

    OpenAIRE

    Wentzel, Johannes Frederik

    2014-01-01

    Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for ...

  18. Surface co-expression of two different PfEMP1 antigens on single Plasmodium falciparum-infected erythrocytes facilitates binding to ICAM1 and PECAM1

    DEFF Research Database (Denmark)

    Joergensen, Louise; Bengtsson, Dominique C; Bengtsson, Anja

    2010-01-01

    The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var...... gene is expressed per cell at a time. We measured var mRNA transcript levels by real-time Q-PCR, analysed var gene transcripts by single-cell FISH and directly compared these with PfEMP1 antigen surface expression and cytoadhesion in three different antibody-selected P. falciparum 3D7 sub-lines using......-line was found to bind both CD31/PECAM1 and CD54/ICAM1 and to adhere twice as efficiently to human endothelial cells, compared to infected cells having only one PfEMP1 variant on the surface. These new results on PfEMP1 antigen expression indicate that a re-evaluation of the molecular mechanisms involved in P...

  19. Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Lisa Gerner

    2016-09-01

    Full Text Available Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2 has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 ‘apo’, CaMKK2 (165-501 in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, “Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2” [1].

  20. A subpopulation of dopaminergic neurons co-expresses serotonin in ventral mesencephalic cultures but not after intrastriatal transplantation in a rat model of Parkinsons disease

    DEFF Research Database (Denmark)

    Di Santo, Stefano; Seiler, Stefanie; Ducray, Angélique

    2017-01-01

    Cell replacement therapy is a promising avenue into the investigation and treatment of Parkinson’s disease (PD) and in some cases significant long-term motor improvements have been demonstrated. The main source of donor tissue is the human fetal ventral mesencephalon (VM), which consists...

  1. Identification of co-expression gene networks, regulatory genes and pathways for obesity based on adipose tissue RNA Sequencing in a porcine model

    DEFF Research Database (Denmark)

    Kogelman, Lisette; Cirera Salicio, Susanna; Zhernakova, Daria V.

    2014-01-01

    Background Obesity is a complex metabolic condition in strong association with various diseases, like type 2 diabetes, resulting in major public health and economic implications. Obesity is the result of environmental and genetic factors and their interactions, including genome-wide genetic...... (modules). Additionally, regulator genes were detected using Lemon-Tree algorithms. Results WGCNA revealed five modules which were strongly correlated with at least one obesity-related phenotype (correlations ranging from -0.54 to 0.72, P ... the association between obesity and other diseases, like osteoporosis (osteoclast differentiation, P = 1.4E-7), and immune-related complications (e.g. Natural killer cell mediated cytotoxity, P = 3.8E-5; B cell receptor signaling pathway, P = 7.2E-5). Lemon-Tree identified three potential regulator genes, using...

  2. Identification of co-expression gene networks, regulatory genes and pathways for obesity based on adipose tissue RNA Sequencing in a porcine model

    NARCIS (Netherlands)

    Kogelman, Lisette J. A.; Cirera, Susanna; Zhernakova, Daria V.; Fredholm, Merete; Franke, Lude; Kadarmideen, Haja N.

    2014-01-01

    Background: Obesity is a complex metabolic condition in strong association with various diseases, like type 2 diabetes, resulting in major public health and economic implications. Obesity is the result of environmental and genetic factors and their interactions, including genome-wide genetic

  3. Efficient expression and characterization of a cold-active endo-1, 4-β-glucanase from Citrobacter farmeri by co-expression of Myxococcus xanthus protein S

    Directory of Open Access Journals (Sweden)

    Xi Bai

    2016-11-01

    Conclusions: The ProS-EglC is promising in application of various biotechnological processes because of its cold-active characterizations. This study also suggests a useful strategy for the expression of foreign proteins in E. coli using a ProS tag.

  4. Transient co-expression for fast and high-yield production of antibodies with human-like N-glycans in plants

    National Research Council Canada - National Science Library

    Vézina, Louis-P; Faye, Loïc; Lerouge, Patrice; D'Aoust, Marc-André; Marquet-Blouin, Estelle; Burel, Carole; Lavoie, Pierre-Olivier; Bardor, Muriel; Gomord, Véronique

    2009-01-01

    Plant-based transient expression is potentially the most rapid and cost-efficient system for the production of recombinant pharmaceutical proteins, but safety concerns associated with plant-specific N...

  5. Cytokines, IgG subclasses and costimulation in a mouse model of thyroid autoimmunity induced by injection of fibroblasts co-expressing MHC class II and thyroid autoantigens

    Science.gov (United States)

    Yan, X-M; Guo, J; Pichurin, P; Tanaka, K; Jaume, J C; Rapoport, B; Mclachlan, S M

    2000-01-01

    AKR/N mice injected with fibroblasts expressing MHC class II (RT4.15HP cells) and the TSH receptor (TSHR) develop antibodies similar to those in Graves' disease. We were unable to analyse the subclass of these antibodies because of unexpectedly high non-specific binding by ELISA or flow cytometry. The non-specific binding reflected generalized immune activation which occurred even when the fibroblasts did not express the TSHR. However, the IgG subclasses were determined for thyroid peroxidase (TPO) antibodies induced using TPO-expressing RT4.14HP cells and found to be IgG2a > IgG1. This Th1 pattern is consistent with spontaneous secretion of interferon-gamma (but not IL-4 or IL-10) by splenocytes from injected mice. The Th1 bias was related to fibroblast injection because conventional immunization of the same mouse strain with purified TPO and adjuvant induced a Th2 response (IgG1 >> IgG2a). Further, untransfected fibroblasts themselves induced powerful, non-specific proliferative responses when used as antigen-presenting cells (APC) in vitro. Flow cytometry revealed that the RT4.15HP fibroblasts (and TSHR- and TPO-transfected derivatives) expressed B7-1. Unexpected constitutive expression of this key molecule may bypass the requirement for up-regulation of other costimulatory molecules involved in T cell stimulation. Our data support the concept that RT4.15HP fibroblasts present the TSHR (or TPO), at least for initiating the immune response. However, the accompanying generalized immune stimulation creates difficulties for analysis of TSHR-specific T and B lymphocytes. On the other hand, extension of the model to TPO, an easier antigen to study, will facilitate analysis of murine T cell responses likely to resemble those in human thyroid autoimmunity. PMID:11091271

  6. Transient Co-Expression of Post-Transcriptional Gene Silencing Suppressors for Increased in Planta Expression of a Recombinant Anthrax Receptor Fusion Protein

    OpenAIRE

    Kittipong Rattanaporn; Maclean, James M.; McDonald, Karen A.; Junxing Chen; Lucas Arzola

    2011-01-01

    Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein,...

  7. A gene co-expression network in whole blood of schizophrenia patients is independent of antipsychotic-use and enriched for brain-expressed genes

    DEFF Research Database (Denmark)

    de Jong, Simone; Boks, Marco P M; Fuller, Tova F

    2012-01-01

    of these robustly defined disease modules is significantly enriched with brain-expressed genes and with genetic variants that were implicated in a GWAS study, which could imply a causal role in schizophrenia etiology. The most highly connected intramodular hub gene in this module (ABCF1), is located in......Despite large-scale genome-wide association studies (GWAS), the underlying genes for schizophrenia are largely unknown. Additional approaches are therefore required to identify the genetic background of this disorder. Here we report findings from a large gene expression study in peripheral blood...... of schizophrenia patients and controls. We applied a systems biology approach to genome-wide expression data from whole blood of 92 medicated and 29 antipsychotic-free schizophrenia patients and 118 healthy controls. We show that gene expression profiling in whole blood can identify twelve large gene co...

  8. Co-expression of bacterial aspartate kinase and adenylylsulfate reductase genes substantially increases sulfur amino acid levels in transgenic alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Tong, Zongyong; Xie, Can; Ma, Lei; Liu, Liping; Jin, Yongsheng; Dong, Jiangli; Wang, Tao

    2014-01-01

    Alfalfa (Medicago sativa L.) is one of the most important forage crops used to feed livestock, such as cattle and sheep, and the sulfur amino acid (SAA) content of alfalfa is used as an index of its nutritional value. Aspartate kinase (AK) catalyzes the phosphorylation of aspartate to Asp-phosphate, the first step in the aspartate family biosynthesis pathway, and adenylylsulfate reductase (APR) catalyzes the conversion of activated sulfate to sulfite, providing reduced sulfur for the synthesis of cysteine, methionine, and other essential metabolites and secondary compounds. To reduce the feedback inhibition of other metabolites, we cloned bacterial AK and APR genes, modified AK, and introduced them into alfalfa. Compared to the wild-type alfalfa, the content of cysteine increased by 30% and that of methionine increased substantially by 60%. In addition, a substantial increase in the abundance of essential amino acids (EAAs), such as aspartate and lysine, was found. The results also indicated a close connection between amino acid metabolism and the tricarboxylic acid (TCA) cycle. The total amino acid content and the forage biomass tested showed no significant changes in the transgenic plants. This approach provides a new method for increasing SAAs and allows for the development of new genetically modified crops with enhanced nutritional value.

  9. Co-Expression of Plexin-B1 and Met in Human Breast and Ovary Tumours Enhances the Risk of Progression

    Directory of Open Access Journals (Sweden)

    Guido Valente

    2009-01-01

    Full Text Available Background: Plex-B1, the receptor of Sema4D, has been implicated in tumour growth, angiogenesis and metastasis. The binding of Sema4D to Plex-B1 can trigger the activation of Met tyrosine kinase, thereby promoting cell dissociation and invasive growth. We tested the hypothesis that the expression of Plex-B1, either alone or in association with Met, can be of predictive value for tumour progression.

  10. Grote rol voor agrarische natuur

    NARCIS (Netherlands)

    Visser, A.J.

    2000-01-01

    In het kader van het BIOM-project (Biologische landbouw, Innovatie en Omschakeling) is varkenshouder Van der Groes in Haps (Noord-Brabant) omgeschakeld, en een schoolvoorbeeld geworden van multifunctionele landbouw, met fokzeugen, mestvarkens, melkgeiten, minicamping en boerderijwinkel. Op alle

  11. Genomic structure of the human mitochondrial chaperonin genes: Hsp60 and Hsp10 are localised head to head on chromosome 2 separated by a bidirectional promoter

    DEFF Research Database (Denmark)

    Hansen, J.J.; Bross, P.; Westergaard, M.

    2003-01-01

    at the air-liquid interface on devitalized dermis, we were able to establish a multilayered epithelium showing histologic similarities to that evolved from native keratinocytes or keratinocytes transduced with the reporter gene encoding enhanced green fluorescent protein. Receptor-modified epidermal tissue......Platelet-derived growth factor is a major proliferative and migratory stimulus for connective tissue cells during the initiation of skin repair processes. In response to injury, locally produced platelet-derived growth factor is secreted by a diversity of cutaneous cell types whereas target...... activity is confined to cells of mesenchymal origin, e.g. dermal fibroblasts and smooth muscle cells. Although epidermal cells contribute to cutaneous platelet-derived growth factor activity by their ample capacity to secrete platelet-derived growth factor ligand, normal epidermal keratinocytes...

  12. ORF Alignment: NC_002945 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ... Structure Of The Mycobacterium Tuberculosis Chaperonin ... 10 Tetradecamer pdb|1P3H|L Chain L, C...rystal Structure Of ... The Mycobacterium Tuberculosis Chaperonin 10 ... ... ... Tetradecamer pdb|1P3H|K Chain K, Crystal Structure Of ... The Mycobacterium Tuberculosis Chaper...onin 10 ... Tetradecamer pdb|1P3H|J Chain J, Crystal Structure Of ... The Mycobacterium Tuberculosis...e Of ... The Mycobacterium Tuberculosis Chaperonin 10 ... Tetradecamer pdb|1P3H|H Chain H, Cry

  13. ORF Alignment: NC_002755 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ... Structure Of The Mycobacterium Tuberculosis Chaperonin ... 10 Tetradecamer pdb|1P3H|L Chain L, C...rystal Structure Of ... The Mycobacterium Tuberculosis Chaperonin 10 ... ... ... Tetradecamer pdb|1P3H|K Chain K, Crystal Structure Of ... The Mycobacterium Tuberculosis Chaper...onin 10 ... Tetradecamer pdb|1P3H|J Chain J, Crystal Structure Of ... The Mycobacterium Tuberculosis...e Of ... The Mycobacterium Tuberculosis Chaperonin 10 ... Tetradecamer pdb|1P3H|H Chain H, Cry

  14. ORF Alignment: NC_000962 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ... Structure Of The Mycobacterium Tuberculosis Chaperonin ... 10 Tetradecamer pdb|1P3H|L Chain L, C...rystal Structure Of ... The Mycobacterium Tuberculosis Chaperonin 10 ... ... ... Tetradecamer pdb|1P3H|K Chain K, Crystal Structure Of ... The Mycobacterium Tuberculosis Chaper...onin 10 ... Tetradecamer pdb|1P3H|J Chain J, Crystal Structure Of ... The Mycobacterium Tuberculosis...e Of ... The Mycobacterium Tuberculosis Chaperonin 10 ... Tetradecamer pdb|1P3H|H Chain H, Cry

  15. Enhancement of solubility in Escherichia coli and purification of an aminotransferase from Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1

    Directory of Open Access Journals (Sweden)

    Hartinger Doris

    2010-08-01

    Full Text Available Abstract Background Fumonisin B1 is a cancerogenic mycotoxin produced by Fusarium verticillioides and other fungi. Sphingopyxis sp. MTA144 can degrade fumonisin B1, and a key enzyme in the catabolic pathway is an aminotransferase which removes the C2-amino group from hydrolyzed fumonisin B1. In order to study this aminotransferase with respect to a possible future application in enzymatic fumonisin detoxification, we attempted expression of the corresponding fumI gene in E. coli and purification of the enzyme. Since the aminotransferase initially accumulated in inclusion bodies, we compared the effects of induction level, host strain, expression temperature, solubility enhancers and a fusion partner on enzyme solubility and activity. Results When expressed from a T7 promoter at 30°C, the aminotransferase accumulated invariably in inclusion bodies in DE3 lysogens of the E. coli strains BL21, HMS174, Rosetta 2, Origami 2, or Rosetta-gami. Omission of the isopropyl-beta-D-thiogalactopyranoside (IPTG used for induction caused a reduction of expression level, but no enhancement of solubility. Likewise, protein production but not solubility correlated with the IPTG concentration in E. coli Tuner(DE3. Addition of the solubility enhancers betaine and sorbitol or the co-enzyme pyridoxal phosphate showed no effect. Maltose-binding protein, used as an N-terminal fusion partner, promoted solubility at 30°C or less, but not at 37°C. Low enzyme activity and subsequent aggregation in the course of purification and cleavage indicated that the soluble fusion protein contained incorrectly folded aminotransferase. Expression in E. coli ArcticExpress(DE3, which co-expresses two cold-adapted chaperonins, at 11°C finally resulted in production of appreciable amounts of active enzyme. Since His tag-mediated affinity purification from this strain was hindered by co-elution of chaperonin, two steps of chromatography with optimized imidazole concentration in the

  16. Primary central nervous system diffuse large B-cell lymphoma shows an activated B-cell-like phenotype with co-expression of C-MYC, BCL-2, and BCL-6.

    Science.gov (United States)

    Li, Xiaomei; Huang, Ying; Bi, Chengfeng; Yuan, Ji; He, Hong; Zhang, Hong; Yu, QiuBo; Fu, Kai; Li, Dan

    2017-06-01

    Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma, whose main prognostic factor is closely related to germinal center B-cell-like subtype (GCB- DLBCL) or activated B-cell-like type (non-GCB-DLBCL). The most common type of primary central nervous system lymphoma is diffuse large B-cell type with poor prognosis and the reason is unclear. This study aims to stratify primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) according to the cell-of-origin (COO) and to investigate the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53, further to elucidate the reason why primary central nervous system diffuse large B-cell lymphoma possesses a poor clinical outcome as well. Nineteen cases of primary central nervous system DLBCL were stratified according to immunostaining algorithms of Hans, Choi and Meyer (Tally) and we investigated the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53. The Epstein-Barr virus and Borna disease virus infection were also detected. Among nineteen cases, most (15-17 cases) were assigned to the activated B-cell-like subtype, highly expression of C-MYC (15 cases, 78.9%), BCL-2 (10 cases, 52.6%), BCL-6 (15 cases, 78.9%). Unfortunately, two cases were positive for PD-L1 while PD-L2 was not expressed in any case. Two cases infected with BDV but no one infected with EBV. In conclusion, most primary central nervous system DLBCLs show an activated B-cell-like subtype characteristic and have multiple expressions of C-MYC, BCL-2, BCL-6 protein, these features might be significant factor to predict the outcome and guide treatment of PCNS-DLBCLs. Copyright © 2017 Elsevier GmbH. All rights reserved.

  17. Co-expression of α9β1 integrin and VEGF-D confers lymphatic metastatic ability to a human breast cancer cell line MDA-MB-468LN.

    Directory of Open Access Journals (Sweden)

    Mousumi Majumder

    Full Text Available INTRODUCTION AND OBJECTIVES: Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN, a variant of the MDA-MB-468GFP (468GFP human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9β1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1 differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9β1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy; (2 differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3 stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice. RESULTS: A comparison of expression of cyclo-oxygenase (COX-2 (a VEGF-C/-D inducer, VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9β1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9β1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9β1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic metastasis in nude mice was completely abrogated by stable knock-down of either VEGF-D or α9 in 468LN cells. CONCLUSION: Differential capacity for VEGF-D production and α9β1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype.

  18. Influence of simulated microgravity on clock genes expression rhythmicity and underlying blood circulating miRNAs-mRNA co-expression regulatory mechanism in C57BL/6J mice

    Science.gov (United States)

    Lv, Ke; Qu, Lina

    Purpose: It is vital for astronauts to maintain the optimal alertness and neurobehavioral function. Among various factors that exist in the space flight and long-duration mission environment, gravity changes may probably an essential environmental factor to interfere with internal circadian rhythms homeostasis and sleep quality, but the underlying mechanism is unclear. Mammals' biological clock is controlled by the suprachiasmatic nucleus (SCN), and peripheral organs adjust their own rhythmicity with the central signals. Nevertheless the mechanism underlying this synchronizition process is still unknown. microRNAs (miRNAs) are about 19˜22nt long regulatory RNAs that serve as critical modulators of post-transcriptional gene regulation. Recently, circulating miRNAs were found to have the regulatory role between cells and peripheral tissues, besides its function inside the cells. This study aims to investigate the regulatory signal transduction role of miRNAs between SCN and peripheral biological clock effecter tissues and to further decipher the mechanism of circadian disturbance under microgravity. Method: Firstly, based on the assumption that severe alterations in the expression of genes known to be involved in circadian rhythms may affect the expression of other genes, the labeled cDNA from liver and suprachiasmatic nucleus (SCN) of clock-knockout mice and control mice in different time points were cohybridized to microarrays. The fold change exceeding 2 (FC>2) was used to identify genes with altered expression levels in the knockout mice compared with control mice. Secondly, male C57BL/6J mice at 8 weeks of age were individually caged and acclimatized to the laboratory conditions (12h light/dark cycle) before being used for continuous core body temperature and activity monitoring. The mice were individually caged and tail suspended using a strip of adhesive surgical tape attached to a chain hanging from a pulley. Peripheral blood and liver tissues collection were consecutively performed. Blood samples and liver tissues were collected from tail-suspended and control mice under LD 12:12h and DD conditions during the 12th, 13th and 14th testing days at 4h intervals. Melatonin and corticosterone in mice plasma at different time points were assayed. NIH-3T3 cells were plated in culture dish for 22h before the experiment. For ground-based simulation of weightlessness, the medium was exchanged with DMEM containing 50% horse serum to synchronization, after 2 h, this medium was replaced with DMEM and 10% FBS. Then, at various time point (0, 6, 12, 18, 24, 30, 36, 42, 48h), cells were cultured on the roating clinostat at 30r/min. Total RNA was extracted from liver and NIH-3T3 cells and subsequently reverse-transcribed. The SYBR green I real-time quantitative PCR system was conducted to examine the mRNA expression level of clock, bmal1, per1, per2, cry1 and cry2 in mice and NIH-3T3 cells, respectively. Paired comparisons of the circadian genes expression between period, peak values, amplitude and mesor (midline estimating statistic of rhythm) were examined for evidence of circadian variation using Chronos-Fit software in mice and Cosine analyses in NIH-3T3 cells. Statistical analysis: All numerical data were expressed as the mean ± standard deviation (SD). Statistical differences among groups were analyzed by one-way analysis of variance (ANOVA) to determine time points differences in the study parameters. Statistical differences between two groups were determined by the Student's t test. Results: (1) Circadian rhythm of clock and bmal1 mRNA expression was found in each testing day with similar peak phase in both tail suspension group and control group. Compared with control group, tail suspension group showed that the peak phase of clock gene mRNA level advanced approximately 4 hours and the amplitude of bmal1 gene mRNA level significantly reduced at ZT2 and ZT6. (2) The expression of circadian genes in NIH-3T3 cells demonstrated that the maximum and minimum value of mRNA relative expression levels of clock and bmal1 during clinorotation were both found approximately at the time points 6h and 18h, respectively. The period length of experimental group was about 16h longer than control group. The peak phase and peak time of clock and bmal1 with simulated weightlessness group were ahead of control group. (3) At the Zeitgeber time 2 (ZT2), we found that 23 miRNAs in the SCN and 60 miRNAs in liver were significantly altered on the basis of an adjusted FC>2 among 611 miRNAs. At the ZT14, 23 miRNAs in the SCN and 57 miRNAs in liver were altered compared with the control group (FC>2). (a) Effects of clock knock out altered expression of miRNA. We analyzed the miRNA profile in SCN and liver of clock knonck out and WT mouse at two different time points using miRNA microarray. Of these, miR-122,miR-144, miR-210 and miR-669b at ZT2, miR-200a, miR-200b, miR-429, miR-455, miR-669d and miR-96 at ZT14 were both changed in SCN and liver, respectively. Interestingly, the miR-122, a tissue specific miRNA of liver was also changed in SCN at ZT2. (b) Effects of light altered expression of miRNA: Light is an important environmental factors to regulate circadian genes expression. In clock mutant mice, all altered miRNAs except miR-144 were down-regulated in SCN while up-regulated in liver at ZT14 compared to ZT2. Interestingly, the miRNAs expression profiling in SCN and liver were opposite of WT mice at ZT14 compared to ZT2. (c) Effects of clock mutant on mRNA expression: To test whether the alteration in expression of miRNAs correlates with the gene expression pattern, cDNA microarray of SCN were assayed. The results revealed that the expression of nearly 1285 genes was altered substantially with at least 1 fold change absolute in the absence of clock. Among these altered genes, we chose the mRNAs with at least 4 fold changes to further study. Only 23 genes were altered in clock knockout compared with WT at ZT2, but 67 genes at ZT14. (d) Effects of light on mRNA expression. To evaluate the light effecting on genes expression in SCN, the cDNA microarrays in SCN at ZT2 and ZT14 were tested. 21 genes were over expression and 12 genes were down regulation ZT14 compared with ZT2 in WT. The number of altered genes in clock-/- mice was 67. (e) Direct interaction between altered miRNAs and mRNAs. To identify the interaction between regulatory miRNAs and altered mRNAs in the absence of clock, we predicted the target genes of miRNAs by TargetScan. The genes both the target genes of miRNAs and altered in cDNA microarray were unravelled. The exploration of functional interaction between miRNAs and clock genes mRNA is ongoing. Conclusion: Taken together, these results indicate that ground-based simulated weightlessness could alter the molecular biological rhythm patterns, which may preliminarily present the biological regulatory mechanism of circadian rhythm systems under spaceflight-related gravity. The potential underlying functional miRNAs could serve as targets to interfere with for interaction between central and peripheral circadian organs under simulated microgravity. This preliminary study may facilitate the exploration of circadian rhythm characteristics in space and the detailed process of signal transduction and circadian gene regulation. Key words: circadian rhythms, tail-suspension, simulated microgravity, clock genes, miRNAs Acknowledgments: This study was supported by the National Basic Research Program of China (Grant NO. 2011CB707704) and the Foundation of State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center (Grant NO. SMFA13B02, SMFA09A06 and SMFA12B05).

  19. Co-Expression of Tyrosine Hydroxylase and GTP Cyclohydrolase I in Arginine Vasopressin-Synthesizing Neurons of the Human Supraoptic Nucleus Demonstrated by Laser Microdissection and Real-Time PCR.

    NARCIS (Netherlands)

    Kontostavlaki, D.P.; Sluijs, J.A.; Unmehopa, U.A.; Huitinga, I.; Hol, E.M.; Swaab, D.F.

    2006-01-01

    Tyrosine hydroxylase (TH), the first and limiting enzyme for catecholamine synthesis, has been identified immunohistochemically (IHC) in human neurosecretory neurons where it is found to colocalize with vasopressin (AVP) or oxytocin. TH expression shows striking interindividual variability and

  20. Prognostic value of the MicroRNA regulators Dicer and Drosha in non-small-cell lung cancer: co-expression of Drosha and miR-126 predicts poor survival

    OpenAIRE

    Lønvik, Kenneth; Sørbye, Sveinung Wergeland; Nilsen, Marit Nina; Paulssen, Ruth H

    2014-01-01

    Background Dicer and Drosha are important enzymes for processing microRNAs. Recent studies have exhibited possible links between expression of different miRNAs, levels of miRNA processing enzymes, and cancer prognosis. We have investigated the prognostic impact of Dicer and Drosha and their correlation with miR-126 expression in a large cohort of non-small cell lung cancer (NSCLC) patients. We aimed to find patient groups within the cohort that might have an advantage of receiving adjunctive ...

  1. A novel patatin-like protein from cotton plant, GhPat1, is co-expressed with GhLox1 during Xanthomonas campestris-mediated hypersensitive cell death.

    Science.gov (United States)

    Cacas, Jean-Luc; Marmey, Philippe; Montillet, Jean-Luc; Sayegh-Alhamdia, Majd; Jalloul, Aida; Rojas-Mendoza, Ana; Clérivet, Alain; Nicole, Michel

    2009-01-01

    In cotton plant, Xanthomonas-induced hypersensitive response (HR) is accompanied by a lipid peroxidation process involving a 9-lipoxygenase (LOX), GhLox1. Initiation of this oxidative metabolism implies the release of the LOX substrates, or polyunsaturated fatty acids. Since patatin-like proteins (PLPs) are likely candidates for mediating the latter step, we searched for genes encoding such enzymes, identified and cloned one of them that we named GhPat1. Biochemical and molecular studies showed that GhPat1 expression was up-regulated during the incompatible interaction, prior to the onset of the corresponding galactolipase activity and cell death symptoms in tissues. Protein sequence analysis and modelling also revealed that GhPat1 catalytic amino acids and fold were conserved across plant PLPs. Based on these results and our previous work (Jalloul et al. in Plant J 32:1-12, 2002), a role for GhPat1, in synergy with GhLox1, during HR-specific lipid peroxidation is discussed.

  2. SSX cancer testis antigens are expressed in most multiple myeloma patients: co-expression of SSX1, 2, 4, and 5 correlates with adverse prognosis and high frequencies of SSX-positive PCs.

    NARCIS (Netherlands)

    Taylor, B.J.; Reiman, T.; Pittman, J.A.; Keats, J.J.; Bruijn, D.R.H. de; Mant, M.J.; Belch, A.R.; Pilarski, L.M.

    2005-01-01

    Cancer testis antigens (CTAs) are tumor-specific antigens that may be useful targets for cancer vaccines. Here, CTA expression was examined in multiple myeloma (MM), a B-cell cancer characterized by malignant plasma cells (PCs) in the bone marrow (BM), and monoclonal gammopathy of undetermined

  3. Identification of differential co-expressed gene networks in early rheumatoid arthritis achieving sustained drug-free remission after treatment with a tocilizumab-based or methotrexate-based strategy

    NARCIS (Netherlands)

    Teitsma, Xavier M.; Jacobs, Johannes W.G.; Mokry, Michal; Borm, Michelle E A; Pethö-Schramm, Attila; van Laar, Jacob M.; Bijlsma, Johannes W.J.; Lafeber, Floris P.J.

    2017-01-01

    Background: Methotrexate is endorsed to be used as first-line treatment in rheumatoid arthritis (RA). However, a large proportion of patients need additional treatment with a biological disease-modifying anti-rheumatic drug (DMARD) to adequately suppress their disease activity. A better

  4. Cells co-expressing luteinising hormone and thyroid-stimulating hormone are present in the ovine pituitary pars distalis but not the pars tuberalis: implications for the control of endogenous circannual rhythms of prolactin.

    Science.gov (United States)

    Hodson, David J; Townsend, Julie; Tortonese, Domingo J

    2013-01-01

    A mammalian circannual pacemaker responsible for regulating the seasonal pattern of prolactin has been recently described in sheep. This pacemaker resides within the pars tuberalis, an area of the pituitary gland that densely expresses melatonin receptors. However, the chemical identity of the cell type which acts as the pacemaker remains elusive. Mathematical-modelling approaches have established that this cell must be responsive to the static melatonin signal as well as prolactin negative feedback. Considering that in sheep the gonadotroph is the only cell in the pars tuberalis which expresses the prolactin receptor, and that in other photoperiodic species the thyrotroph is the only cell expressing the melatonin receptor in this tissue, a cell type which expresses both proteins would fulfil the theoretical criteria of a circannual pacemaker. Pituitary glands were obtained from female sheep under short days (breeding season) and long days (non-breeding season) and double immunofluorescent staining was conducted to determine the prevalence of bi-hormonal cells in the pars distalis and pars tuberalis using specific antibodies to luteinising hormone-β and thyroid-stimulating hormone-β. The results reveal that whilst such a bihormonal cell is clearly present in the pars distalis and constitute 4% of the gonadotroph population in this region, the same cell type is completely absent from the pars tuberalis even though LH gonadotrophs are abundantly expressed. Based on these findings, together with existing data, we are able to propose an alternative model where the gonadotroph itself is controlled indirectly by neighbouring melatonin responsive cells, allowing it to act as a pacemaker. Copyright © 2013 S. Karger AG, Basel.

  5. A high-yield co-expression system for the purification of an intact drs2p-cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate

    DEFF Research Database (Denmark)

    Azouaoui, Hassina; Montigny, Cédric; Ash, Miriam-Rose

    2014-01-01

    a fraction that mainly contained a 1∶1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated...... was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity......P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic...

  6. The extremely divergent maternally- and paternally-transmitted mitochondrial genomes are co-expressed in somatic tissues of two freshwater mussel species with doubly uniparental inheritance of mtDNA.

    Science.gov (United States)

    Breton, Sophie; Bouvet, Karim; Auclair, Gabrielle; Ghazal, Stéphanie; Sietman, Bernard E; Johnson, Nathan; Bettinazzi, Stefano; Stewart, Donald T; Guerra, Davide

    2017-01-01

    Freshwater mussel species with doubly uniparental inheritance (DUI) of mtDNA are unique because they are naturally heteroplasmic for two extremely divergent mtDNAs with ~50% amino acid differences for protein-coding genes. The paternally-transmitted mtDNA (or M mtDNA) clearly functions in sperm in these species, but it is still unknown whether it is transcribed when present in male or female soma. In the present study, we used PCR and RT-PCR to detect the presence and expression of the M mtDNA in male and female somatic and gonadal tissues of the freshwater mussel species Venustaconcha ellipsiformis and Utterbackia peninsularis (Unionidae). This is the first study demonstrating that the M mtDNA is transcribed not only in male gonads, but also in male and female soma in freshwater mussels with DUI. Because of the potentially deleterious nature of heteroplasmy, we suggest the existence of different mechanisms in DUI species to deal with this possibly harmful situation, such as silencing mechanisms for the M mtDNA at the transcriptional, post-transcriptional and/or post-translational levels. These hypotheses will necessitate additional studies in distantly-related DUI species that could possess different mechanisms of action to deal with heteroplasmy.

  7. First characterisation of plasmid-mediated quinolone resistance-qnrS1 co-expressed bla CTX-M-15 and bla DHA-1 genes in clinical strain of Morganella morganii recovered from a Tunisian Intensive Care Unit

    Directory of Open Access Journals (Sweden)

    S Mahrouki

    2012-01-01

    Full Text Available Purpose: Aim of this study was to show the emergence of the qnr genes among fluoroquinolone-resistant, AMPC and ESBL (extended-spectrum-beta-lactamase co-producing Morganella morganii isolate. Materials and Methods: A multi resistant Morganella morganii SM12012 isolate was recovered from pus from a patient hospitalized in the intensive care unit at the Military hospital, Tunisia. Antibiotic susceptibility was tested with the agar disk diffusion method according to Clinical and Laboratory Standards Institute guidelines. ESBLs were detected using a standard double-disk synergy test. The characterization of beta-lactamases and associated resistance genes were performed by isoelectric focusing, polymerase chain reaction and nucleotide sequencing. Results: The antimicrobial susceptibility testing showed the high resistance to penicillins, cephalosporins (MICs: 64-512 μg/ml and fluoroquinolones (MICs: 32-512 μg/ml. But M. morganii SM12012 isolate remained susceptible to carbapenems (MICs: 4-<0.25 μg/ml. The double-disk synergy test confirmed the phenotype of extended-spectrum β-lactamases (ESBLs. Three identical β-lactamases with pI values of 6.5, 7.8 and superior to 8.6 were detected after isoelectric focusing analysis. These β-lactamases genes can be successfully transferred by the conjugative plasmid. Molecular analysis demonstrated the co-production of bla DHA-1, bla CTX-M-15 and qnrS1 genes on the same plasmid. The detection of an associated chromosomal quinolone resistance revealed the presence of a parC mutation at codon 80 (Ser80-lle80. Conclusion: This is the first report in Tunisia of nosocomial infection due to the production of CTX-M-15 and DHA-1 β-lactamases in M. morganii isolate with the association of quinolone plasmid resistance. The incidence of these strains invites continuous monitoring of such multidrug-resistant strains and the further study of their epidemiologic evolution.

  8. Opsins in Limulus eyes: characterization of three visible light-sensitive opsins unique to and co-expressed in median eye photoreceptors and a peropsin/RGR that is expressed in all eyes

    OpenAIRE

    Battelle, Barbara-Anne; Kempler, Karen E.; Saraf, Spencer R.; Marten, Catherine E.; Dugger, Donald R.; Speiser, Daniel I.; Oakley, Todd H.

    2015-01-01

    The eyes of the horseshoe crab Limulus polyphemus have long been used for studies of basic mechanisms of vision, and the structure and physiology of Limulus photoreceptors have been examined in detail. Less is known about the opsins Limulus photoreceptors express. We previously characterized a UV opsin (LpUVOps1) that is expressed in all three types of Limulus eyes (lateral compound eyes, median ocelli and larval eyes) and three visible light-sensitive rhabdomeric opsins (LpOps1, -2 and -5) t...

  9. Co-expression of three MEP pathway genes and geraniol 10-hydroxylase in internal phloem parenchyma of Catharanthus roseus implicates multicellular translocation of intermediates during the biosynthesis of monoterpene indole alkaloids and isoprenoid-derived primary metabolites.

    Science.gov (United States)

    Burlat, Vincent; Oudin, Audrey; Courtois, Martine; Rideau, Marc; St-Pierre, Benoit

    2004-04-01

    In higher plants, isopentenyl diphosphate (IPP) is synthesised both from the plastidic 2-C-methyl-d-erythritol 4-phosphate (MEP) and from the cytosolic mevalonate (MVA) pathways. Primary metabolites, such as phytol group of chlorophylls, carotenoids and the plant hormones abscisic acid (ABA) and gibberellins (GAs) are derived directly from the MEP pathway. Many secondary metabolites, such as monoterpene indole alkaloids (MIAs) in Catharanthus roseus, are also synthesised from this source of IPP. Using Northern blot and in situ hybridisation experiments, we show that three MEP pathway genes (1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MECS)) and the gene encoding geraniol 10-hydroxylase (G10H), a cytochrome P450 monooxygenase involved in the first committed step in the formation of iridoid monoterpenoids display identical cell-specific expression patterns. The co-localisation of these four transcripts to internal phloem parenchyma of young aerial organs of C. roseus adds a new level of complexity to the multicellular nature of MIA biosynthesis. We predict the translocation of pathway intermediates from the internal phloem parenchyma to the epidermis and, ultimately, to laticifers and idioblasts during MIA biosynthesis. Similarly, the translocation of intermediates from the phloem parenchyma is probably also required during the biosynthesis of hormones and photosynthetic primary metabolites derived from the MEP pathway.

  10. A high-yield co-expression system for the purification of an intact drs2p-cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate

    DEFF Research Database (Denmark)

    Azouaoui, Hassina; Montigny, Cédric; Ash, Miriam-Rose

    2014-01-01

    P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic......, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼1-2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover...... was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity...

  11. Dicty_cDB: VSE435 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available . 209 4e-53 S26582( S26582 ) chaperonin hsp60 - maize &Z12114_1(Z12114|pid:no... 209 4e-53 S20875( S20875...S20875 ;S34488;S19844) chaperonin hsp60 precursor - maize ... 209 4e-53 L21007_1( L21007 |pid:none) Corn

  12. Dicty_cDB: FC-AO06 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available . 211 3e-53 S26583( S26583 ) chaperonin hsp60 - maize &Z12115_1(Z12115|pid:no... 211 3e-53 ( P29185 ).... 209 8e-53 S26582( S26582 ) chaperonin hsp60 - maize &Z12114_1(Z12114|pid:no... 209 8e-53 AC161864_25(

  13. Dicty_cDB: SLB321 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available . 242 6e-63 S26582( S26582 ) chaperonin hsp60 - maize &Z12114_1(Z12114|pid:no... 242 6e-63 S20875( S20875...S20875 ;S34488;S19844) chaperonin hsp60 precursor - maize ... 241 1e-62 L21007_1( L21007 |pid:none) Corn

  14. Dicty_cDB: VSE277 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available . 111 7e-24 S26582( S26582 ) chaperonin hsp60 - maize &Z12114_1(Z12114|pid:no... 111 7e-24 S20875( S20875...S20875 ;S34488;S19844) chaperonin hsp60 precursor - maize ... 111 7e-24 AF065609_1( AF065609 |pid:none) Toxoplasma

  15. Science Cafes: Engaging graduate students one drink at a time!

    Science.gov (United States)

    Schiebel, H.; Chen, R. F.

    2016-02-01

    Science Cafes are events that take place in casual settings (pubs, coffeehouses) that are typically open to a broad audience and feature engaging conversations with scientists about particular topics. Science Cafes are a grassroots movement and exist on an international scale with a common goal of engaging broad audiences in informal scientific discussions. Graduate Students for Ocean Education (GrOE), funded by COSEE OCEAN (Center for Ocean Science Education Excellence—Ocean Communities in Science Education And social Networks), has taken this model and honed in on a specific audience: graduate students. Through monthly Science Cafes with varying themes (ocean acidification to remote sensing), GrOE has engaged over two hundred graduate students throughout New England. While attendance at the Science Cafes is consistent, the presence and engagement of graduate students on the GrOE Facebook page is now growing, a trend attributed to having face-to-face contact with scientists and other graduate students.

  16. ORF Sequence: NC_003281 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003281 gi|17557043 >gi|17557043|ref|NP_498707.1| copper (CU) Chaperonin, functi...onal, complements yeast atx1 mutant (7.6 kD) (cuc-1) [Caenorhabditis elegans] MTQYVFEMGMTCNGCANAARKVLGKLGEDKIKIDDINVETKKITVTTDLPASDVLEALKKTGKEIKQLQ

  17. Genetics Home Reference: McKusick-Kaufman syndrome

    Science.gov (United States)

    ... AD, Barr M, Biesecker LG. Mutation of a gene encoding a putative chaperonin causes McKusick-Kaufman syndrome. Nat Genet. ... Features What are genome editing and CRISPR-Cas9? What is direct-to-consumer ...

  18. Beyond Dichotomies of Victimization versus Agency

    DEFF Research Database (Denmark)

    Spanger, Marlene

    2016-01-01

    A new growing literature that merges from the scholarly fields of migration and sex work questions the concepts of ‘sex work’ and ‘prostitution’. Instead, this literature (Cabezas 2006, Fair 2007, Casas 2010, Spanger 2012 and 2013, Mai 2013, Groes-Gren 2013 etc.) argues in different ways that the...

  19. Dicty_cDB: Contig-U15272-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available .0 2 ( AB242233 ) Lettuce yellows phytoplasma groES, groEL, amp, na... 34 7.0 2 ( AB124810 ) Paulownia witch...Clover phyllody phytoplasma immunodominant membra... 34 7.4 2 ( DQ515794 ) Paulow

  20. Comparing permanent tooth sizes of Nigerians and American Negroes

    African Journals Online (AJOL)

    sizes of Nigerians have been that of Mack P.J3 while those on. American Negroes have been by Richardson and Malhotra in. 1975 where normal values were established for male and female. American Negroes. The aim of this paper is to compare the mesiodistal crown dimensions of Nigerians and American Ne- groes.

  1. The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

    Science.gov (United States)

    Jiang, Ming; Zhang, Haoran; Pfeifer, Blaine A.

    2013-01-01

    , chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation. Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach. PMID:23354010

  2. The logic, experimental steps, and potential of heterologous natural product biosynthesis featuring the complex antibiotic erythromycin A produced through E. coli.

    Science.gov (United States)

    Jiang, Ming; Zhang, Haoran; Pfeifer, Blaine A

    2013-01-13

    , chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation. Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

  3. Co-expression et caractérisation fonctionnelle d’un transporteur de lipides (une « flippase ») de la levure S. cerevisiae : l’ATPase P4 Drs2p, en complexe avec sa sous-unité associée Cdc50p

    OpenAIRE

    Jacquot, Aurore

    2012-01-01

    Trans-Golgi membranes and plasma membranes of eukaryotic cells are asymmetric, with their cytosolic leaflet enriched in aminophospholipids (APLs: phosphatidylserine and phosphatidylethanolamine). Dissipation of this asymmetry is involved in many (patho)physiological processes. P4 ATPases are prime candidates for APL transport and for maintaining asymmetry across membranes. In addition, yeast deleted for P4 ATPases display membrane trafficking defects. Besides, CDC50 proteins have been shown t...

  4. Inhibition of the Secretory pathway by Foot-and-Mouth disease virus 2BC protein is reproduced by co-expression of 2B with 2C, and the site of inhibition is determined by the subcellular location of 2C

    DEFF Research Database (Denmark)

    Moffat, Katy; Knox, Caroline; Howell, Gareth

    2007-01-01

    immune responses in vivo. Foot-and-mouth disease virus (FMDV), another picornavirus, can cause persistent infection of ruminants, suggesting it too may inhibit immune responses. Endoplasmic reticulum (ER)-to-Golgi apparatus transport of proteins is blocked by the FMDV 2BC protein. The observation that 2...... blocked in FMDV-infected cells. The block could be reconstituted by coexpression of 2B and 2C, showing that processing of 2BC did not compromise the ability of FMDV to slow secretion. Under these conditions, 2C was located to the Golgi apparatus, and the block in transport also occurred in the Golgi...... apparatus. Interestingly, the block in transport could be redirected to the ER when 2B was coexpressed with a 2C protein fused to an ER retention element. Thus, for FMDV a block in secretion is dependent on both 2B and 2C, with the latter determining the site of the block....

  5. Los complejos Chaperonina(MSP63inducen anticuerpos de reacciones cruzadas, bactericidas y opsonofagocítica.

    Directory of Open Access Journals (Sweden)

    Juan Marzoa

    2009-08-01

    Full Text Available Alteration of the native structure of antigens can lead to the loss of protective epitopes. Our previous results showed that separation of the meningococcal outer membrane proteins in native conditions revealed the existence of protein complexes that could be relevant for the development of new vaccine formulations. The aim of this work was to analyse the immunogenic characteristics of a highly conserved 700 kDa chaperonin complex (CxChap detected and purified by using high resolution clear native electrophoresis. Analysis of the anti-CxChap serum by Western-blotting revealed the presence of antibodies against the MSP63 but also against the macrophage infectivity potentiator-like protein (MIP, which is coopurified with the chaperonin complex. Antibodies raised by immunisation with CxChap chaperonin complex show bactericidal and opsonophagocytic activity.

  6. Dicty_cDB: Contig-U15138-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 6e-59 AB379695_1( AB379695 |pid:none) Enterobacteriaceae bacterium Epu-T... 230 6e-59 (Q7UM97) RecName: Ful...) RecName: Full=60 kDa chaperonin 2; AltName: Full=Protei... 228 3e-58 AB379694_1( AB379694 |pid:none) Enterobacteriaceae

  7. Dicty_cDB: Contig-U06612-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 4 |pid:none) Ehrlichia ruminantium strain Kumm1... 45 6e-04 AJ302157_1( AJ302157 |pid:none) Enterobacteria...ceae sp. JM965 parti... 45 6e-04 (Q8Y1P9) RecName: Full=10 kDa chaperonin; AltName:

  8. Analysis of Chaperone Complexes by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

    NARCIS (Netherlands)

    Geels, R.B.J.

    2008-01-01

    Investigation of methodologies for analyses of noncovalently bound protein assemblies using Fourier transformation ion cyclotron resonance mass spectrometry (FT-ICR-MS) and quadrupole Time-of-Flight (qToF) mass spectrometry. Specifically, the co-chaperonins GroEL and gp31 are used to perform

  9. 12038_2017_9728_MOESM1_ESM.docx

    Indian Academy of Sciences (India)

    jOyOuS jOsEpH

    Protein phosphatase (PP)2A, Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X ... CPN60-2. RNA polymerase. sigma-24 subunit. Unstable. PKHD-type. hydroxylase. Type III secretion. component protein. Chaperonin. CPN60-2. Root, Stable. NAD- ...

  10. Effects of solar UV-B radiation on canopy structure of Ulva communities from southern Spain

    NARCIS (Netherlands)

    Bischof, K; Peralta, G; Krabs, G; van de Poll, WH; Perez-Llorens, JL; Breeman, AM

    2002-01-01

    Within the sheltered creeks of Cadiz bay, Ulva thalli form extended mat-like canopies. The effect of solar ultraviolet radiation on photosynthetic activity, the composition of photosynthetic and xanthophyll cycle pigments, and the amount of RubisCO, chaperonin 60 (CPN 60), and the induction of DNA

  11. Growth, symbiotic, and proteomics studies of soybean ...

    African Journals Online (AJOL)

    ajl10

    2012-10-16

    Oct 16, 2012 ... Interestingly, GroEL protein is involved in nif gene regulation in B. japonicum. It was proposed that one or more of chaperonin proteins assembled with nitrogenase and assisting the proper folding of nitrogenase complex, and finally link to nodulation and nitrogen fixation efficiency of bacteria (Lund,. 2009).

  12. SwissProt search result: AK061410 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061410 006-306-B08 (P35635) 60 kDa chaperonin (Protein Cpn60) (groEL protein) (Immunoreactive... protein Bb65) (Immunoreactive protein Bb63) (Heat shock protein 60) (HSP 60) CH60_BARBA 2e-63 ...

  13. SwissProt search result: AK060117 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060117 006-308-E09 (P35635) 60 kDa chaperonin (Protein Cpn60) (groEL protein) (Immunoreactive... protein Bb65) (Immunoreactive protein Bb63) (Heat shock protein 60) (HSP 60) CH60_BARBA 2e-71 ...

  14. SwissProt search result: AK120503 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK120503 J013122K17 (P35635) 60 kDa chaperonin (Protein Cpn60) (groEL protein) (Immunoreactive... protein Bb65) (Immunoreactive protein Bb63) (Heat shock protein 60) (HSP 60) CH60_BARBA 1e-141 ...

  15. SwissProt search result: AK060773 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060773 001-033-B08 (P35635) 60 kDa chaperonin (Protein Cpn60) (groEL protein) (Immunoreactive... protein Bb65) (Immunoreactive protein Bb63) (Heat shock protein 60) (HSP 60) CH60_BARBA 2e-53 ...

  16. SwissProt search result: AK069617 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069617 J023019K12 (P35635) 60 kDa chaperonin (Protein Cpn60) (groEL protein) (Immunoreactive... protein Bb65) (Immunoreactive protein Bb63) (Heat shock protein 60) (HSP 60) CH60_BARBA 0.0 ...

  17. SwissProt search result: AK070127 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK070127 J023045O03 (P35635) 60 kDa chaperonin (Protein Cpn60) (groEL protein) (Immunoreactive... protein Bb65) (Immunoreactive protein Bb63) (Heat shock protein 60) (HSP 60) CH60_BARBA 1e-157 ...

  18. SwissProt search result: AK073999 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK073999 J033073C08 (P35635) 60 kDa chaperonin (Protein Cpn60) (groEL protein) (Immunoreactive... protein Bb65) (Immunoreactive protein Bb63) (Heat shock protein 60) (HSP 60) CH60_BARBA 1e-177 ...

  19. SwissProt search result: AK070603 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK070603 J023059D06 (P35635) 60 kDa chaperonin (Protein Cpn60) (groEL protein) (Immunoreactive... protein Bb65) (Immunoreactive protein Bb63) (Heat shock protein 60) (HSP 60) CH60_BARBA 1e-150 ...

  20. SwissProt search result: AK101537 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101537 J033048E22 (P35635) 60 kDa chaperonin (Protein Cpn60) (groEL protein) (Immunoreactive... protein Bb65) (Immunoreactive protein Bb63) (Heat shock protein 60) (HSP 60) CH60_BARBA 1e-151 ...