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Sample records for generates protein variants

  1. Method of generating ploynucleotides encoding enhanced folding variants

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    Bradbury, Andrew M.; Kiss, Csaba; Waldo, Geoffrey S.

    2017-05-02

    The invention provides directed evolution methods for improving the folding, solubility and stability (including thermostability) characteristics of polypeptides. In one aspect, the invention provides a method for generating folding and stability-enhanced variants of proteins, including but not limited to fluorescent proteins, chromophoric proteins and enzymes. In another aspect, the invention provides methods for generating thermostable variants of a target protein or polypeptide via an internal destabilization baiting strategy. Internally destabilization a protein of interest is achieved by inserting a heterologous, folding-destabilizing sequence (folding interference domain) within DNA encoding the protein of interest, evolving the protein sequences adjacent to the heterologous insertion to overcome the destabilization (using any number of mutagenesis methods), thereby creating a library of variants. The variants in the library are expressed, and those with enhanced folding characteristics selected.

  2. Generation and Efficacy Evaluation of a Recombinant Pseudorabies Virus Variant Expressing the E2 Protein of Classical Swine Fever Virus in Pigs.

    Science.gov (United States)

    Wang, Yimin; Yuan, Jin; Cong, Xin; Qin, Hua-Yang; Wang, Chun-Hua; Li, Yongfeng; Li, Su; Luo, Yuzi; Sun, Yuan; Qiu, Hua-Ji

    2015-10-01

    Classical swine fever (CSF) is an economically important infectious disease of pigs caused by classical swine fever virus (CSFV). Pseudorabies (PR), which is caused by pseudorabies virus (PRV), is another important infectious disease of pigs and other animals. Coinfections of pigs with PRV and CSFV occur occasionally in the field. The modified live vaccine Bartha-K61 strain has played an important role in the control of PR in many countries, including China. Since late 2011, however, increasing PR outbreaks caused by an emerging PRV variant have been reported in Bartha-K61-vaccinated swine populations on many farms in China. Previously, we generated a gE/gI-deleted PRV (rPRVTJ-delgE) based on this PRV variant, which was shown to be safe and can provide rapid and complete protection against lethal challenge with the PRV variant in pigs. Here, we generated a new recombinant PRV variant expressing the E2 gene of CSFV (rPRVTJ-delgE/gI-E2) and evaluated its immunogenicity and efficacy in pigs. The results showed that rPRVTJ-delgE/gI-E2 was safe for pigs, induced detectable anti-PRV and anti-CSFV neutralizing antibodies, and provided complete protection against the lethal challenge with either the PRV TJ strain or the CSFV Shimen strain. The data indicate that rPRVTJ-delgE/gI-E2 is a promising candidate bivalent vaccine against PRV and CSFV coinfections.

  3. Alternative splicing of CARMA2/CARD14 transcripts generates protein variants with differential effect on NF-κB activation and endoplasmic reticulum stress-induced cell death.

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    Scudiero, Ivan; Zotti, Tiziana; Ferravante, Angela; Vessichelli, Mariangela; Vito, Pasquale; Stilo, Romania

    2011-12-01

    The caspase recruitment domain (CARD)-containing proteins CARMA1-3 share high degree of sequence, structure and functional homology. Whereas CARMA1 and CARMA3 have been identified as crucial components of signal transduction pathways that lead to activation of NF-κB transcription factor, little is known about the function of CARMA2. Here we report the identification of two splice variants of CARMA2. One transcript, named CARMA2short (CARMA2sh), is predicted to encode for a CARMA2 polypeptide containing the CARD, coiled coil, and a PDZ domains, but lacking the SH3 and the GuK domains. The second variant, CARMA2cardless (CARMA2cl), encodes for a polypeptide lacking the CARD domain and containing only a portion of the coiled coil domain and a linker region. Expression analysis confirmed the presence of the CARMA2 alternatively spliced transcripts in both human cell lines and tissues. Fluorescence microscopy data show that both splice variants localize in the cytosol. Biochemical experiments indicate that CARMA2sh interacts with TRAF2 and activates NF-κB in a TRAF2-dependent manner. Finally, CARMA2sh variant protects cells from apoptosis induced by different stimuli. Taken together, these results demonstrate that multiple transcripts encoding several CARMA2 isoforms exist in vivo and regulate NF-κB activation and apoptosis.

  4. Nuclear variants of bone morphogenetic proteins

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    Meinhart Christopher A

    2010-03-01

    Full Text Available Abstract Background Bone morphogenetic proteins (BMPs contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts. Results In all three proteins, a bipartite nuclear localization signal (NLS was found to overlap the site at which the proproteins are cleaved to release the mature growth factors from the propeptides. Mutational analyses indicated that the nuclear variants of these three proteins are produced by initiating translation from downstream alternative start codons. The resulting proteins lack N-terminal signal peptides and are therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing in the secretory pathway. Instead, the uncleaved proteins (designated nBmp2, nBmp4, and nGdf5 containing the intact NLSs are translocated to the nucleus. Immunostaining of endogenous nBmp2 in cultured cells demonstrated that the amount of nBmp2 as well as its nuclear/cytoplasmic distribution differs between cells that are in M-phase versus other phases of the cell cycle. Conclusions The observation that nBmp2 localization varies throughout the cell cycle, as well as the conservation of a nuclear localization mechanism among three different BMP family members, suggests that these novel nuclear variants of BMP family proteins play an important functional role in the cell.

  5. Targeted quantitative mass spectrometric immunoassay for human protein variants

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    Nedelkov Dobrin

    2011-04-01

    Full Text Available Abstract Background Post-translational modifications and genetic variations give rise to protein variants that significantly increase the complexity of the human proteome. Modified proteins also play an important role in biological processes. While sandwich immunoassays are routinely used to determine protein concentrations, they are oblivious to protein variants that may serve as biomarkers with better sensitivity and specificity than their wild-type proteins. Mass spectrometry, coupled to immunoaffinity separations, can provide an efficient mean for simultaneous detection and quantification of protein variants. Results Presented here is a mass spectrometric immunoassay method for targeted quantitative proteomics analysis of protein modifications. Cystatin C, a cysteine proteinase inhibitor and a potential marker for several pathological processes, was used as a target analyte. An internal reference standard was incorporated into the assay, serving as a normalization point for cystatin C quantification. The precision, linearity, and recovery characteristics of the assay were established. The new assay was also benchmarked against existing cystatin C ELISA. In application, the assay was utilized to determine the individual concentration of several cystatin C variants across a cohort of samples, demonstrating the ability to fully quantify individual forms of post-translationally modified proteins. Conclusions The mass spectrometric immunoassays can find use in quantifying specific protein modifications, either as a part of a specific protein biomarker discovery/rediscovery effort to delineate the role of these variants in the onset of the disease, progression, and response to therapy, or in a more systematic study to delineate and understand human protein diversity.

  6. Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

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    Anamika Krishanpal

    2009-12-01

    Full Text Available Abstract Background Protein kinases are involved in diverse spectrum of cellular processes. Availability of draft version of the human genomic data in the year 2001 enabled recognition of repertoire of protein kinases. However, over the years the human genomic data is being refined and the current release of human genomic data has helped us to recognize a larger repertoire of over 900 human protein kinases represented mainly by splice variants. Results Many of these identified protein kinases are alternatively spliced products. Interestingly, some of the human kinase splice variants appear to be significantly diverged in terms of their functional properties as represented by incorporation or absence of one or more domains. Many sets of protein kinase splice variants have substantially different domain organization and in a few sets of splice variants kinase domains belong to different subfamilies of kinases suggesting potential participation in different signal transduction pathways. Conclusions Addition or deletion of a domain between splice variants of multi-domain kinases appears to be a means of generating differences in the functional features of otherwise similar kinases. It is intriguing that marked sequence diversity within the catalytic regions of some of the splice variant kinases result in kinases belonging to different subfamilies. These human kinase splice variants with different functions might contribute to diversity of eukaryotic cellular signaling.

  7. Progressive Concept Evaluation Method for Automatically Generated Concept Variants

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    Woldemichael Dereje Engida

    2014-07-01

    Full Text Available Conceptual design is one of the most critical and important phases of design process with least computer support system. Conceptual design support tool (CDST is a conceptual design support system developed to automatically generate concepts for each subfunction in functional structure. The automated concept generation process results in large number of concept variants which require a thorough evaluation process to select the best design. To address this, a progressive concept evaluation technique consisting of absolute comparison, concept screening and weighted decision matrix using analytical hierarchy process (AHP is proposed to eliminate infeasible concepts at each stage. The software implementation of the proposed method is demonstrated.

  8. Modeling of protein-anion exchange resin interaction for the human growth hormone charge variants.

    Science.gov (United States)

    Lapelosa, Mauro; Patapoff, Thomas W; Zarraga, Isidro E

    2015-12-01

    Modeling ion exchange chromatography (IEC) behavior has generated significant interest because of the wide use of IEC as an analytical technique as well as a preparative protein purification process; indeed there is a need for better understanding of what drives the unique behavior of protein charge variants. We hypothesize that a complex protein molecule, which contains both hydrophobic and charged moieties, would interact strongly with an in silico designed resin through charged electrostatic patches on the surface of the protein. In the present work, variants of recombinant human growth hormone that mimic naturally-occurring deamidation products were produced and characterized in silico. The study included these four variants: rhGH, N149D, N152D, and N149D/N152D. Poisson-Boltzmann calculations were used to determine surface electrostatic potential. Metropolis Monte Carlo simulations were carried out with the resulting variants to simulate IEC systems, examining the free energy of the interaction of the protein with an in silico anion exchange column represented by polylysine polypeptide. The results show that the charge variants have different average binding energies and the free energy of interaction can be used to predict the retention time for the different variants.

  9. Phenotypic comparisons of consensus variants versus laboratory resurrections of Precambrian proteins.

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    Risso, Valeria A; Gavira, Jose A; Gaucher, Eric A; Sanchez-Ruiz, Jose M

    2014-06-01

    Consensus-sequence engineering has generated protein variants with enhanced stability, and sometimes, with modulated biological function. Consensus mutations are often interpreted as the introduction of ancestral amino acid residues. However, the precise relationship between consensus engineering and ancestral protein resurrection is not fully understood. Here, we report the properties of proteins encoded by consensus sequences derived from a multiple sequence alignment of extant, class A β-lactamases, as compared with the properties of ancient Precambrian β-lactamases resurrected in the laboratory. These comparisons considered primary sequence, secondary, and tertiary structure, as well as stability and catalysis against different antibiotics. Out of the three consensus variants generated, one could not be expressed and purified (likely due to misfolding and/or low stability) and only one displayed substantial stability having substrate promiscuity, although to a lower extent than ancient β-lactamases. These results: (i) highlight the phenotypic differences between consensus variants and laboratory resurrections of ancestral proteins; (ii) question interpretations of consensus proteins as phenotypic proxies of ancestral proteins; and (iii) support the notion that ancient proteins provide a robust approach toward the preparation of protein variants having large numbers of mutational changes while possessing unique biomolecular properties. © 2014 Wiley Periodicals, Inc.

  10. Identification of novel splice variants of Adhesion G protein-coupled receptors.

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    Bjarnadóttir, Thóra K; Geirardsdóttir, Kristín; Ingemansson, Malena; Mirza, Majd A I; Fredriksson, Robert; Schiöth, Helgi B

    2007-01-31

    Alternative splicing is an important mechanism to generate proteome diversity in higher eukaryotic organisms. We searched for splice variants of the human Adhesion family of G protein-coupled receptors (GPCRs) using mRNA sequences and expressed sequence tags. The results presented here describe 53 human splice variants among the 33 Adhesion GPCRs. Many of these variants appear to be coding for "functional" proteins (29) while the others are seemingly "non-functional" (24). Novel functional splice variants were found for: CD97, CELR3, EMR2, EMR3, GPR56, GPR110, GPR112-GPR114, GPR116, GPR123-GPR126, GPR133, HE6, and LEC1-LEC3. Splice variants for GPR116, GPR125, GPR126, and HE6 were found conserved in other species. Several of the functional splice variants lack one or more of the functional domains that are found in the N-termini of these receptors. These functional domains are likely to affect ligand binding or interaction with other proteins and these novel splice variants may have important roles for the specificity of interactions between these receptors and extracellular molecules. Another type of splice variants found here lacks a GPCR proteolytic site (GPS). The GPS domain has been shown to be essential for the proteolytic cleavage of the receptors N-termini and for cellular surface expression. We suggest that these alternative splice variants may be crucial for the function of the receptors while the seemingly non-functional splice variants may be a part of a regulative mechanism.

  11. GenOVa: a computer program to generate orientational variants

    OpenAIRE

    Cayron, Cyril

    2007-01-01

    A computer program called GenOVa, written in Python, calculates the orientational variants, the operators (special types of misorientations between variants) and the composition table associated with a groupoid structure. The variants can be represented by three-dimensional shapes or by pole figures.

  12. PLZF-RAR[alpha] fusion proteins generated from the variant t(11; 17)(q23; q21) translocation in acute promyelocytic leukemia inhibit ligand-dependent transactivation of wild-type retinoic acid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhu; Chen, Sai-Juan; Wang, Zhen-Yi (Shanghai Second Medical Univ. (China)); Guidez, F.; Rousselot, P.; Agadir, A.; Degos, L.; Chomienne, C. (Laboratoire de Biologie Cellulaire Hematopoietique, Paris (France)); Zelent, A. (Institute for Cancer Research, London (United Kingdom)); Waxman, S. (Mount Sinai Medical Center, New York, NY (United States))

    1994-02-01

    Recently, the authors described a recurrent variant translocation, t(11;17)(q23;q21), in acute promyelocytic leukemia (APL) which juxtaposes PLZF, a gene encoding a zinc finger protein, to RARA, encoding retinoic acid receptor [alpha] (RAR[alpha]). They have now cloned cDNAs encoding PLZF-RAR[alpha] chimeric proteins and studied their transactivating activities. In transient-expression assays, both the PLZF(A)-RAR[alpha] and PLZF(B)-RAR[alpha] fusion proteins like the PML-RAR[alpha] protein resulting from the well-known t(15;17) translocation in APL, antagonized endogenous and transfected wild-type RAR[alpha] in the presence of retinoic acid. Cotransfection assays showed that a significant repression of RAR[alpha] transactivation activity was obtained even with a very low PLZF-RAR[alpha]-expressing plasmid concentration. A [open quotes]dominant negative[close quotes] effect was observed with vectors expressing RAR[alpha] and retinoid X receptor [alpha] (RXR[alpha]). These abnormal transactivation properties observed in retinoic acid-sensitive myeloid cells strongly implicate the PLZF-RAR[alpha] fusion proteins in the molecular pathogenesis of APL.

  13. Extensive libraries of gene truncation variants generated by in vitro transposition.

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    Morelli, Aleardo; Cabezas, Yari; Mills, Lauren J; Seelig, Burckhard

    2017-01-26

    The detailed analysis of the impact of deletions on proteins or nucleic acids can reveal important functional regions and lead to variants with improved macromolecular properties. We present a method to generate large libraries of mutants with deletions of varying length that are randomly distributed throughout a given gene. This technique facilitates the identification of crucial sequence regions in nucleic acids or proteins. The approach utilizes in vitro transposition to generate 5' and 3' fragment sub-libraries of a given gene, which are then randomly recombined to yield a final library comprising both terminal and internal deletions. The method is easy to implement and can generate libraries in three to four days. We used this approach to produce a library of >9000 random deletion mutants of an artificial RNA ligase enzyme representing 32% of all possible deletions. The quality of the library was assessed by next-generation sequencing and detailed bioinformatics analysis. Finally, we subjected this library to in vitro selection and obtained fully functional variants with deletions of up to 18 amino acids of the parental enzyme that had been 95 amino acids in length.

  14. Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants.

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    Karkov, Hanne Sophie; Krogh, Berit Olsen; Woo, James; Parimal, Siddharth; Ahmadian, Haleh; Cramer, Steven M

    2015-11-01

    In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules.

  15. Delineation of concentration ranges and longitudinal changes of human plasma protein variants.

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    Olgica Trenchevska

    Full Text Available Human protein diversity arises as a result of alternative splicing, single nucleotide polymorphisms (SNPs and posttranslational modifications. Because of these processes, each protein can exists as multiple variants in vivo. Tailored strategies are needed to study these protein variants and understand their role in health and disease. In this work we utilized quantitative mass spectrometric immunoassays to determine the protein variants concentration of beta-2-microglobulin, cystatin C, retinol binding protein, and transthyretin, in a population of 500 healthy individuals. Additionally, we determined the longitudinal concentration changes for the protein variants from four individuals over a 6 month period. Along with the native forms of the four proteins, 13 posttranslationally modified variants and 7 SNP-derived variants were detected and their concentration determined. Correlations of the variants concentration with geographical origin, gender, and age of the individuals were also examined. This work represents an important step toward building a catalog of protein variants concentrations and examining their longitudinal changes.

  16. αIIbβ3 variants defined by next-generation sequencing: Predicting variants likely to cause Glanzmann thrombasthenia

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    Buitrago, Lorena; Rendon, Augusto; Liang, Yupu; Simeoni, Ilenia; Negri, Ana; Filizola, Marta; Ouwehand, Willem H.; Coller, Barry S.; Alessi, Marie-Christine; Ballmaier, Matthias; Bariana, Tadbir; Bellissimo, Daniel; Bertoli, Marta; Bray, Paul; Bury, Loredana; Carrell, Robin; Cattaneo, Marco; Collins, Peter; French, Deborah; Favier, Remi; Freson, Kathleen; Furie, Bruce; Germeshausen, Manuela; Ghevaert, Cedric; Gomez, Keith; Goodeve, Anne; Gresele, Paolo; Guerrero, Jose; Hampshire, Dan J.; Hadinnapola, Charaka; Heemskerk, Johan; Henskens, Yvonne; Hill, Marian; Hogg, Nancy; Johnsen, Jill; Kahr, Walter; Kerr, Ron; Kunishima, Shinji; Laffan, Michael; Natwani, Amit; Neerman-Arbez, Marguerite; Nurden, Paquita; Nurden, Alan; Ormiston, Mark; Othman, Maha; Ouwehand, Willem; Perry, David; Vilk, Shoshana Ravel; Reitsma, Pieter; Rondina, Matthew; Simeoni, Ilenia; Smethurst, Peter; Stephens, Jonathan; Stevenson, William; Szkotak, Artur; Turro, Ernest; Van Geet, Christel; Vries, Minka; Ward, June; Waye, John; Westbury, Sarah; Whiteheart, Sidney; Wilcox, David; Zhang, Bi

    2015-01-01

    Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin αIIbβ3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising ∼32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting ∼11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting ∼9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of αIIbβ3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. αIIb P176H and β3 C547G severely reduced αIIbβ3 expression, whereas αIIb P943A partially reduced αIIbβ3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69–98% sensitivity in detecting GT mutations, between 27% and 71% of the novel αIIb or β3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on αIIbβ3 and highlight the challenges in predicting the clinical significance of novel missense variants. PMID:25827233

  17. TDP-43 protein variants as biomarkers in amyotrophic lateral sclerosis.

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    Williams, Stephanie M; Khan, Galam; Harris, Brent T; Ravits, John; Sierks, Michael R

    2017-01-25

    TDP-43 aggregates accumulate in individuals affected by amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases, representing potential diagnostic and therapeutic targets. Using an atomic force microscopy based biopanning protocol developed in our lab, we previously isolated 23 TDP-43 reactive antibody fragments with preference for human ALS brain tissue relative to frontotemporal dementia, a related neurodegeneration, and healthy samples from phage-displayed single chain antibody fragment (scFv) libraries. Here we further characterize the binding specificity of these different scFvs and identify which ones have promise for detecting ALS biomarkers in human brain tissue and plasma samples. We developed a sensitive capture ELISA for detection of different disease related TDP-43 variants using the scFvs identified from the ALS biopanning. We show that a wide variety of disease selective TDP-43 variants are present in ALS as the scFvs show different reactivity profiles amongst the ALS cases. When assaying individual human brain tissue cases, three scFvs (ALS-TDP6, ALS-TDP10 and ALS-TDP14) reacted with all the ALS cases and 12 others reacted with the majority of the ALS cases, and none of the scFvs reacted with any control samples. When assaying individual human plasma samples, 9 different scFvs reacted with all the sporadic ALS samples and again none of them reacted with any control samples. These 9 different scFvs had different patterns of reactivity with plasma samples obtained from chromosome 9 open reading frame 72 (c9orf72) cases indicating that these familial ALS genetic variants may display different TDP-43 pathology than sporadic ALS cases. These results indicated that a range of disease specific TDP-43 variants are generated in ALS patients with different variants being generated in sporadic and familial cases. We show that a small panel of scFvs recognizing different TDP-43 variants can generate a neuropathological and plasma biomarker

  18. A protein-truncating R179X variant in RNF186 confers protection against ulcerative colitis

    NARCIS (Netherlands)

    Rivas, Manuel A.; Graham, Daniel; Sulem, Patrick; Stevens, Christine; Desch, A. Nicole; Goyette, Philippe; Gudbjartsson, Daniel; Jonsdottir, Ingileif; Thorsteinsdottir, Unnur; Degenhardt, Frauke; Mucha, Soeren; Kurki, Mitja I.; Li, Dalin; D'Amato, Mauro; Annese, Vito; Vermeire, Severine; Weersma, Rinse K.; Halfvarson, Jonas; Paavola-Sakki, Paulina; Lappalainen, Maarit; Lek, Monkol; Cummings, Beryl; Tukiainen, Taru; Haritunians, Talin; Halme, Leena; Koskinen, Lotta L. E.; Ananthakrishnan, Ashwin N.; Luo, Yang; Heap, Graham A.; Visschedijk, Marijn C.; MacArthur, Daniel G.; Neale, Benjamin M.; Ahmad, Tariq; Anderson, Carl A.; Brant, Steven R.; Duerr, Richard H.; Silverberg, Mark S.; Cho, Judy H.; Palotie, Aarno; Saavalainen, Paivi; Kontula, Kimmo; Farkkila, Martti; McGovern, Dermot P. B.; Franke, Andre; Stefansson, Kari; Rioux, John D.; Xavier, Ramnik J.; Daly, Mark J.

    2016-01-01

    Protein-truncating variants protective against human disease provide in vivo validation of therapeutic targets. Here we used targeted sequencing to conduct a search for protein-truncating variants conferring protection against inflammatory bowel disease exploiting knowledge of common variants associ

  19. Next generation sequencing reveals the antibiotic resistant variants in the genome of Pseudomonas aeruginosa.

    Science.gov (United States)

    Ramanathan, Babu; Jindal, Hassan Mahmood; Le, Cheng Foh; Gudimella, Ranganath; Anwar, Arif; Razali, Rozaimi; Poole-Johnson, Johan; Manikam, Rishya; Sekaran, Shamala Devi

    2017-01-01

    Rapid progress in next generation sequencing and allied computational tools have aided in identification of single nucleotide variants in genomes of several organisms. In the present study, we have investigated single nucleotide polymorphism (SNP) in ten multi-antibiotic resistant Pseudomonas aeruginosa clinical isolates. All the draft genomes were submitted to Rapid Annotations using Subsystems Technology (RAST) web server and the predicted protein sequences were used for comparison. Non-synonymous single nucleotide polymorphism (nsSNP) found in the clinical isolates compared to the reference genome (PAO1), and the comparison of nsSNPs between antibiotic resistant and susceptible clinical isolates revealed insights into the genome variation. These nsSNPs identified in the multi-drug resistant clinical isolates were found to be altering a single amino acid in several antibiotic resistant genes. We found mutations in genes encoding efflux pump systems, cell wall, DNA replication and genes involved in repair mechanism. In addition, nucleotide deletions in the genome and mutations leading to generation of stop codons were also observed in the antibiotic resistant clinical isolates. Next generation sequencing is a powerful tool to compare the whole genomes and analyse the single base pair variations found within the antibiotic resistant genes. We identified specific mutations within antibiotic resistant genes compared to the susceptible strain of the same bacterial species and these findings may provide insights to understand the role of single nucleotide variants in antibiotic resistance.

  20. Task of generation of variants of subsystems in to the automated hydrometeorological system on basis of morphological synthesis

    OpenAIRE

    Доронина, Юлия Валентиновна

    2011-01-01

    The aspects of generation of variants of subsystems are examined for  the hydrometeorological system. Principles of generation of variants are rotined on the basis of morphological synthesis, statements of genetic algorithm

  1. From Single Variants to Protein Cascades: MULTISCALE MODELING OF SINGLE NUCLEOTIDE VARIANT SETS IN GENETIC DISORDERS.

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    Mueller, Sabine C; Sommer, Björn; Backes, Christina; Haas, Jan; Meder, Benjamin; Meese, Eckart; Keller, Andreas

    2016-01-22

    Understanding the role of genetics in disease has become a central part of medical research. Non-synonymous single nucleotide variants (nsSNVs) in coding regions of human genes frequently lead to pathological phenotypes. Beyond single variations, the individual combination of nsSNVs may add to pathogenic processes. We developed a multiscale pipeline to systematically analyze the existence of quantitative effects of multiple nsSNVs and gene combinations in single individuals on pathogenicity. Based on this pipeline, we detected in a data set of 842 nsSNVs discovered in 76 genes related to cardiomyopathies, associated nsSNV combinations in seven genes present in at least 70% of all 639 patient samples, but not in a control cohort of healthy humans. Structural analyses of these revealed primarily an influence on the protein stability. For amino acid substitutions located at the protein surface, we generally observed a proximity to putative binding pockets. To computationally analyze cumulative effects and their impact, pathogenicity methods are currently being developed. Our approach supports this process, as shown on the example of a cardiac phenotype but can be likewise applied to other diseases such as cancer.

  2. Discontinuous space variant sub-wavelength structures for generating radially polarized light in visible region

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    Ghadyani, Z.; Dmitriev, S.; Lindlein, N.; Leuchs, G.; Rusina, O.; Harder, I.

    2011-08-01

    A discontinuous space variant sub-wavelength dielectric grating is designed and fabricated for generating radially polarized light in visible region (l = 632.8 nm). The design is based on sub-wavelength silicon nitride structures introducing a retardation of p/2 by form birefringence, with space variant orientation of the optical axis. The pattern is divided into concentric ring segments with constant structural parameters, therefore reducing electron-beam writing time significantly. The design avoids the technological challenges encountered in the generation of a continuous space variant grating while maintaining good quality of the resulting polarization mode.

  3. Desired alteration of protein affinities: competitive selection of protein variants using yeast signal transduction machinery.

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    Misato Kaishima

    Full Text Available Molecules that can control protein-protein interactions (PPIs have recently drawn attention as new drug pipeline compounds. Here, we report a technique to screen desirable affinity-altered (affinity-enhanced and affinity-attenuated protein variants. We previously constructed a screening system based on a target protein fused to a mutated G-protein γ subunit (Gγcyto lacking membrane localization ability. This ability, required for signal transmission, is restored by recruiting Gγcyto into the membrane only when the target protein interacts with an artificially membrane-anchored candidate protein, thereby allowing interacting partners (Gγ recruitment system to be searched and identified. In the present study, the Gγ recruitment system was altered by integrating the cytosolic expression of a third protein as a competitor to set a desirable affinity threshold. This enabled the reliable selection of both affinity-enhanced and affinity-attenuated protein variants. The presented approach may facilitate the development of therapeutic proteins that allow the control of PPIs.

  4. MSV3d: database of human MisSense Variants mapped to 3D protein structure.

    Science.gov (United States)

    Luu, Tien-Dao; Rusu, Alin-Mihai; Walter, Vincent; Ripp, Raymond; Moulinier, Luc; Muller, Jean; Toursel, Thierry; Thompson, Julie D; Poch, Olivier; Nguyen, Hoan

    2012-01-01

    The elucidation of the complex relationships linking genotypic and phenotypic variations to protein structure is a major challenge in the post-genomic era. We present MSV3d (Database of human MisSense Variants mapped to 3D protein structure), a new database that contains detailed annotation of missense variants of all human proteins (20 199 proteins). The multi-level characterization includes details of the physico-chemical changes induced by amino acid modification, as well as information related to the conservation of the mutated residue and its position relative to functional features in the available or predicted 3D model. Major releases of the database are automatically generated and updated regularly in line with the dbSNP (database of Single Nucleotide Polymorphism) and SwissVar releases, by exploiting the extensive Décrypthon computational grid resources. The database (http://decrypthon.igbmc.fr/msv3d) is easily accessible through a simple web interface coupled to a powerful query engine and a standard web service. The content is completely or partially downloadable in XML or flat file formats. Database URL: http://decrypthon.igbmc.fr/msv3d.

  5. A Protein Domain and Family Based Approach to Rare Variant Association Analysis

    Science.gov (United States)

    Richardson, Tom G.; Shihab, Hashem A.; Rivas, Manuel A.; McCarthy, Mark I.; Campbell, Colin; Timpson, Nicholas J.; Gaunt, Tom R.

    2016-01-01

    Background It has become common practice to analyse large scale sequencing data with statistical approaches based around the aggregation of rare variants within the same gene. We applied a novel approach to rare variant analysis by collapsing variants together using protein domain and family coordinates, regarded to be a more discrete definition of a biologically functional unit. Methods Using Pfam definitions, we collapsed rare variants (Minor Allele Frequency ≤ 1%) together in three different ways 1) variants within single genomic regions which map to individual protein domains 2) variants within two individual protein domain regions which are predicted to be responsible for a protein-protein interaction 3) all variants within combined regions from multiple genes responsible for coding the same protein domain (i.e. protein families). A conventional collapsing analysis using gene coordinates was also undertaken for comparison. We used UK10K sequence data and investigated associations between regions of variants and lipid traits using the sequence kernel association test (SKAT). Results We observed no strong evidence of association between regions of variants based on Pfam domain definitions and lipid traits. Quantile-Quantile plots illustrated that the overall distributions of p-values from the protein domain analyses were comparable to that of a conventional gene-based approach. Deviations from this distribution suggested that collapsing by either protein domain or gene definitions may be favourable depending on the trait analysed. Conclusion We have collapsed rare variants together using protein domain and family coordinates to present an alternative approach over collapsing across conventionally used gene-based regions. Although no strong evidence of association was detected in these analyses, future studies may still find value in adopting these approaches to detect previously unidentified association signals. PMID:27128313

  6. A Protein Domain and Family Based Approach to Rare Variant Association Analysis.

    Directory of Open Access Journals (Sweden)

    Tom G Richardson

    Full Text Available It has become common practice to analyse large scale sequencing data with statistical approaches based around the aggregation of rare variants within the same gene. We applied a novel approach to rare variant analysis by collapsing variants together using protein domain and family coordinates, regarded to be a more discrete definition of a biologically functional unit.Using Pfam definitions, we collapsed rare variants (Minor Allele Frequency ≤ 1% together in three different ways 1 variants within single genomic regions which map to individual protein domains 2 variants within two individual protein domain regions which are predicted to be responsible for a protein-protein interaction 3 all variants within combined regions from multiple genes responsible for coding the same protein domain (i.e. protein families. A conventional collapsing analysis using gene coordinates was also undertaken for comparison. We used UK10K sequence data and investigated associations between regions of variants and lipid traits using the sequence kernel association test (SKAT.We observed no strong evidence of association between regions of variants based on Pfam domain definitions and lipid traits. Quantile-Quantile plots illustrated that the overall distributions of p-values from the protein domain analyses were comparable to that of a conventional gene-based approach. Deviations from this distribution suggested that collapsing by either protein domain or gene definitions may be favourable depending on the trait analysed.We have collapsed rare variants together using protein domain and family coordinates to present an alternative approach over collapsing across conventionally used gene-based regions. Although no strong evidence of association was detected in these analyses, future studies may still find value in adopting these approaches to detect previously unidentified association signals.

  7. Detection and characterization of two co-infection variant strains of avian orthoreovirus (ARV) in young layer chickens using next-generation sequencing (NGS).

    Science.gov (United States)

    Tang, Yi; Lin, Lin; Sebastian, Aswathy; Lu, Huaguang

    2016-04-19

    Using next-generation sequencing (NGS) for full genomic characterization studies of the newly emerging avian orthoreovirus (ARV) field strains isolated in Pennsylvania poultry, we identified two co-infection ARV variant strains from one ARV isolate obtained from ARV-affected young layer chickens. The de novo assembly of the ARV reads generated 19 contigs of two different ARV variant strains according to 10 genome segments of each ARV strain. The two variants had the same M2 segment. The complete genomes of each of the two variant strains were 23,493 bp in length, and 10 dsRNA segments ranged from 1192 bp (S4) to 3958 bp (L1), encoding 12 viral proteins. Sequence comparison of nucleotide (nt) and amino acid (aa) sequences of all 10 genome segments revealed 58.1-100% and 51.4-100% aa identity between the two variant strains, and 54.3-89.4% and 49.5-98.1% aa identity between the two variants and classic vaccine strains. Phylogenetic analysis revealed a moderate to significant nt sequence divergence between the two variant and ARV reference strains. These findings have demonstrated the first naturally occurring co-infection of two ARV variants in commercial young layer chickens, providing scientific evidence that multiple ARV strains can be simultaneously present in one host species of chickens.

  8. Potential for Improving Potency and Specificity of Reovirus Oncolysis with Next-Generation Reovirus Variants

    Directory of Open Access Journals (Sweden)

    Adil Mohamed

    2015-12-01

    Full Text Available Viruses that specifically replicate in tumor over normal cells offer promising cancer therapies. Oncolytic viruses (OV not only kill the tumor cells directly; they also promote anti-tumor immunotherapeutic responses. Other major advantages of OVs are that they dose-escalate in tumors and can be genetically engineered to enhance potency and specificity. Unmodified wild type reovirus is a propitious OV currently in phase I–III clinical trials. This review summarizes modifications to reovirus that may improve potency and/or specificity during oncolysis. Classical genetics approaches have revealed reovirus variants with improved adaptation towards tumors or with enhanced ability to establish specific steps of virus replication and cell killing among transformed cells. The recent emergence of a reverse genetics system for reovirus has provided novel strategies to fine-tune reovirus proteins or introduce exogenous genes that could promote oncolytic activity. Over the next decade, these findings are likely to generate better-optimized second-generation reovirus vectors and improve the efficacy of oncolytic reotherapy.

  9. Potentiated Hsp104 variants suppress toxicity of diverse neurodegenerative disease-linked proteins

    Directory of Open Access Journals (Sweden)

    Meredith E. Jackrel

    2014-10-01

    Full Text Available Protein misfolding is implicated in numerous lethal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS and Parkinson disease (PD. There are no therapies that reverse these protein-misfolding events. We aim to apply Hsp104, a hexameric AAA+ protein from yeast, to target misfolded conformers for reactivation. Hsp104 solubilizes disordered aggregates and amyloid, but has limited activity against human neurodegenerative disease proteins. Thus, we have previously engineered potentiated Hsp104 variants that suppress aggregation, proteotoxicity and restore proper protein localization of ALS and PD proteins in Saccharomyces cerevisiae, and mitigate neurodegeneration in an animal PD model. Here, we establish that potentiated Hsp104 variants possess broad substrate specificity and, in yeast, suppress toxicity and aggregation induced by wild-type TDP-43, FUS and α-synuclein, as well as missense mutant versions of these proteins that cause neurodegenerative disease. Potentiated Hsp104 variants also rescue toxicity and aggregation of TAF15 but not EWSR1, two RNA-binding proteins with a prion-like domain that are connected with the development of ALS and frontotemporal dementia. Thus, potentiated Hsp104 variants are not entirely non-specific. Indeed, they do not unfold just any natively folded protein. Rather, potentiated Hsp104 variants are finely tuned to unfold proteins bearing short unstructured tracts that are not recognized by wild-type Hsp104. Our studies establish the broad utility of potentiated Hsp104 variants.

  10. Differential protein expression in phenotypic variants of Streptococcus pneumoniae

    NARCIS (Netherlands)

    K. Overweg (Karin); C.D. Pericone; G.G. Verhoef; J.N. Weiser; H.D. Meiring; A.P. de Jong; R. de Groot (Ronald); P.W.M. Hermans (Peter)

    2000-01-01

    textabstractStreptococcus pneumoniae undergoes spontaneous phase variation resulting in opaque and transparent colony forms. Differences in colony opacity correlate with differences in virulence: the transparent variants are more capable of colonizing the nasopharynx, w

  11. A systematic survey of loss-of-function variants in human protein-coding genes

    NARCIS (Netherlands)

    MacArthur, D.G.; Balasubramanian, S.; Frankish, A.; Huang, N.; Morris, J.; Walter, K.; Jostins, L.; Habegger, L.; Pickrell, J.K.; Montgomery, S.B.; Albers, C.A.; Zhang, Z.D.; Conrad, D.F.; Lunter, G.; Zheng, H.; Ayub, Q.; DePristo, M.A.; Banks, E.; Hu, M.; Handsaker, R.E.; Rosenfeld, J.A.; Fromer, M.; Jin, M.; Mu, X.J.; Khurana, E.; Ye, K.; Kay, M.; Saunders, G.I.; Suner, M.M.; Hunt, T.; Barnes, I.H.; Amid, C.; Carvalho-Silva, D.R.; Bignell, A.H.; Snow, C.; Yngvadottir, B.; Bumpstead, S.; Cooper, D.N.; Xue, Y.; Romero, I.G.; Genomes Project, C.; Wang, J.; Li, Y.; Gibbs, R.A.; McCarroll, S.A.; Dermitzakis, E.T.; Pritchard, J.K.; Barrett, J.C.; Harrow, J.; Hurles, M.E.; Gerstein, M.B.; Tyler-Smith, C.

    2012-01-01

    Genome-sequencing studies indicate that all humans carry many genetic variants predicted to cause loss of function (LoF) of protein-coding genes, suggesting unexpected redundancy in the human genome. Here we apply stringent filters to 2951 putative LoF variants obtained from 185 human genomes to det

  12. A protein-truncating R179X variant in RNF186 confers protection against ulcerative colitis

    Science.gov (United States)

    Rivas, Manuel A.; Graham, Daniel; Sulem, Patrick; Stevens, Christine; Desch, A. Nicole; Goyette, Philippe; Gudbjartsson, Daniel; Jonsdottir, Ingileif; Thorsteinsdottir, Unnur; Degenhardt, Frauke; Mucha, Sören; Kurki, Mitja I.; Li, Dalin; D'Amato, Mauro; Annese, Vito; Vermeire, Severine; Weersma, Rinse K.; Halfvarson, Jonas; Paavola-Sakki, Paulina; Lappalainen, Maarit; Lek, Monkol; Cummings, Beryl; Tukiainen, Taru; Haritunians, Talin; Halme, Leena; Koskinen, Lotta L. E.; Ananthakrishnan, Ashwin N.; Luo, Yang; Heap, Graham A.; Visschedijk, Marijn C.; Barrett, J; de Lange, K; Edwards, C; Hart, A; Hawkey, C; Jostins, L; Kennedy, N; Lamb, C; Lee, J; Lees, C; Mansfield, J; Mathew, C; Mowatt, C; Newman, W; Nimmo, E; Parkes, M; Pollard, M; Prescott, N; Randall, J; Rice, D; Satsangi, J; Simmons, A; Tremelling, M; Uhlig, H; Wilson, D; Abraham, C; Achkar, J.P; Bitton, A; Boucher, G; Croitoru, K; Fleshner, P; Glas, J; Kugathasan, S; Limbergen, J.V; Milgrom, R; Proctor, D; Regueiro, M; Schumm, P.L; Sharma, Y; Stempak, J.M; Targan, S.R; Wang, M.H; MacArthur, Daniel G.; Neale, Benjamin M.; Ahmad, Tariq; Anderson, Carl A.; Brant, Steven R.; Duerr, Richard H.; Silverberg, Mark S.; Cho, Judy H; Palotie, Aarno; Saavalainen, Päivi; Kontula, Kimmo; Färkkilä, Martti; McGovern, Dermot P. B.; Franke, Andre; Stefansson, Kari; Rioux, John D.; Xavier, Ramnik J.; Daly, Mark J.; Barrett, J.; de Lane, K.; Edwards, C.; Hart, A.; Hawkey, C.; Jostins, L.; Kennedy, N.; Lamb, C.; Lee, J.; Lees, C.; Mansfield, J.; Mathew, C.; Mowatt, C.; Newman, B.; Nimmo, E.; Parkes, M.; Pollard, M.; Prescott, N.; Randall, J.; Rice, D.; Satsangi, J.; Simmons, A.; Tremelling, M.; Uhlig, H.; Wilson, D.; Abraham, C.; Achkar, J. P.; Bitton, A.; Boucher, G.; Croitoru, K.; Fleshner, P.; Glas, J.; Kugathasan, S.; Limbergen, J. V.; Milgrom, R.; Proctor, D.; Regueiro, M.; Schumm, P. L.; Sharma, Y.; Stempak, J. M.; Targan, S. R.; Wang, M. H.

    2016-01-01

    Protein-truncating variants protective against human disease provide in vivo validation of therapeutic targets. Here we used targeted sequencing to conduct a search for protein-truncating variants conferring protection against inflammatory bowel disease exploiting knowledge of common variants associated with the same disease. Through replication genotyping and imputation we found that a predicted protein-truncating variant (rs36095412, p.R179X, genotyped in 11,148 ulcerative colitis patients and 295,446 controls, MAF=up to 0.78%) in RNF186, a single-exon ring finger E3 ligase with strong colonic expression, protects against ulcerative colitis (overall P=6.89 × 10−7, odds ratio=0.30). We further demonstrate that the truncated protein exhibits reduced expression and altered subcellular localization, suggesting the protective mechanism may reside in the loss of an interaction or function via mislocalization and/or loss of an essential transmembrane domain. PMID:27503255

  13. The next generation of metastatic melanoma: uncovering the genetic variants for anti-BRAF therapy response

    Science.gov (United States)

    Pinto, Rosamaria; De Summa, Simona; Strippoli, Sabino; Pilato, Brunella; Azzariti, Amalia; Guida, Gabriella; Guida, Michele; Tommasi, Stefania

    2016-01-01

    Metastatic melanoma (MM) is a highly aggressive cancer with a median overall survival of 6–9 months, notwithstanding the numerous efforts in development of new therapeutic approaches. To this aim we tested the clinical applicability of the Ion Torrent Personal Genome Machine to simultaneously screen MM patients in order to individuate new or already known SNPs and mutations able to predict the duration of response to BRAF inhibitors. An Ampliseq Custom Panel, including 11 crucial full length genes involved in melanoma carcinogenesis and therapy response pathways, was created and used to analyze 25 MM patients. We reported BRAFV600 and NRASQ61 mutations in 68% and 24% of samples, respectively. Moreover, we more frequently identified the following alterations related to BRAF status: PIK3CAI391M (44%) and KITD737N (36%) mutations, CTLA4T17A (52%), MC1RV60L (32%) and MITFS473A (60%) polymorphisms. Considering the progression free survival (PFS), statistical analyses showed that BRAFV600 patients without any of these more frequent alterations had a higher median PFS. Protein structure changes seem to be due to these variants by in silico analysis. In conclusion, a Next-Generation Sequencing approach with custom panel may provide new information to evaluate tumor-specific therapeutic susceptibility and individual prognosis to improve the care of MM patients. PMID:26863566

  14. The next generation of metastatic melanoma: uncovering the genetic variants for anti-BRAF therapy response.

    Science.gov (United States)

    Pinto, Rosamaria; De Summa, Simona; Strippoli, Sabino; Pilato, Brunella; Azzariti, Amalia; Guida, Gabriella; Guida, Michele; Tommasi, Stefania

    2016-05-01

    Metastatic melanoma (MM) is a highly aggressive cancer with a median overall survival of 6-9 months, notwithstanding the numerous efforts in development of new therapeutic approaches. To this aim we tested the clinical applicability of the Ion Torrent Personal Genome Machine to simultaneously screen MM patients in order to individuate new or already known SNPs and mutations able to predict the duration of response to BRAF inhibitors. An Ampliseq Custom Panel, including 11 crucial full length genes involved in melanoma carcinogenesis and therapy response pathways, was created and used to analyze 25 MM patients. We reported BRAFV600 and NRASQ61 mutations in 68% and 24% of samples, respectively. Moreover, we more frequently identified the following alterations related to BRAF status: PIK3CAI391M (44%) and KITD737N (36%) mutations, CTLA4T17A (52%), MC1RV60L (32%) and MITFS473A (60%) polymorphisms. Considering the progression free survival (PFS), statistical analyses showed that BRAFV600 patients without any of these more frequent alterations had a higher median PFS. Protein structure changes seem to be due to these variants by in silico analysis. In conclusion, a Next-Generation Sequencing approach with custom panel may provide new information to evaluate tumor-specific therapeutic susceptibility and individual prognosis to improve the care of MM patients.

  15. Monomeric red fluorescent protein variants used for imaging studies in different species

    NARCIS (Netherlands)

    Mueller-Taubenberger, Annette; Vos, Michel J.; Boettger, Angelika; Lasi, Margherita; Lai, Frank P. L.; Fischer, Markus; Rottner, Klemens

    2006-01-01

    Fluorescent proteins have proven to be excellent tools for live-cell imaging studies. In addition to green fluorescent protein (GFP) and its variants, recent progress was achieved in the development of monomeric red fluorescent proteins (mRFPs) that show improved properties in respect to maturation

  16. Fe Protein-Independent Substrate Reduction by Nitrogenase MoFe Protein Variants

    Energy Technology Data Exchange (ETDEWEB)

    Danyal, Karamatullah; Rasmussen, Andrew J.; Keable, Stephen M.; Inglet, Boyd S.; Shaw, Sudipta; Zadvornyy, Oleg; Duval, Simon S.; Dean, Dennis R.; Raugei, Simone; Peters, John W.; Seefeldt, Lance C.

    2015-04-21

    The reduction of substrates catalyzed by nitrogenase normally requires nucleotide-dependent Fe protein delivery of electrons to the MoFe protein, which contains the active site FeMo-cofactor. Here, it is reported that independent substitution of three amino acids (ß-98Tyr→His, α-64Tyr→His, and ß-99Phe→His) located between the P cluster and FeMo-cofactor within the MoFe protein endows it with the ability to reduce protons to H2, azide to ammonia, and hydrazine to ammonia without the need for Fe protein or ATP. Instead, electrons can be provided by the low potential reductant polyaminocarboxylate ligated Eu(II) (Em -1.1 to -0.84 V vs NHE). The crystal structure of the ß-98Tyr→His variant MoFe protein was determined, revealing only small changes near the amino acid substitution that affect the solvent structure and immediate vicinity between the P cluster and the FeMo-cofactor, with no global conformational changes observed. Computational normal mode analysis on the nitrogenase complex reveal coupling in the motions of the Fe protein and the region of the MoFe protein with these three amino acids, which suggests a possible mechanism for how Fe protein might communicate deep within the MoFe protein subtle changes that profoundly affect intramolecular electron transfer and substrate reduction. This work was supported by a grant from the National Science Foundation (MCB-1330807) to JWP and LCS. This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences (DE-SC0010687 and DE-SC0010834 to LCS and DRD) and the Division of Chemical Sciences, Geosciences, and Bio-Sciences (SR). The coordinates for the ß-98His MoFe protein were deposited with the Protein Data Bank (PDB 4XPI).

  17. Mitochondrial DNA variant discovery and evaluation in human Cardiomyopathies through next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Michael V Zaragoza

    Full Text Available Mutations in mitochondrial DNA (mtDNA may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We "shotgun" sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300x average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20x while heteroplasmic variants required >200x coverage. Several Sanger "misses" were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease.

  18. Evaluating Variant Calling Tools for Non-Matched Next-Generation Sequencing Data

    Science.gov (United States)

    Sandmann, Sarah; de Graaf, Aniek O.; Karimi, Mohsen; van der Reijden, Bert A.; Hellström-Lindberg, Eva; Jansen, Joop H.; Dugas, Martin

    2017-02-01

    Valid variant calling results are crucial for the use of next-generation sequencing in clinical routine. However, there are numerous variant calling tools that usually differ in algorithms, filtering strategies, recommendations and thus, also in the output. We evaluated eight open-source tools regarding their ability to call single nucleotide variants and short indels with allelic frequencies as low as 1% in non-matched next-generation sequencing data: GATK HaplotypeCaller, Platypus, VarScan, LoFreq, FreeBayes, SNVer, SAMtools and VarDict. We analysed two real datasets from patients with myelodysplastic syndrome, covering 54 Illumina HiSeq samples and 111 Illumina NextSeq samples. Mutations were validated by re-sequencing on the same platform, on a different platform and expert based review. In addition we considered two simulated datasets with varying coverage and error profiles, covering 50 samples each. In all cases an identical target region consisting of 19 genes (42,322 bp) was analysed. Altogether, no tool succeeded in calling all mutations. High sensitivity was always accompanied by low precision. Influence of varying coverages- and background noise on variant calling was generally low. Taking everything into account, VarDict performed best. However, our results indicate that there is a need to improve reproducibility of the results in the context of multithreading.

  19. Evaluating Variant Calling Tools for Non-Matched Next-Generation Sequencing Data

    Science.gov (United States)

    Sandmann, Sarah; de Graaf, Aniek O.; Karimi, Mohsen; van der Reijden, Bert A.; Hellström-Lindberg, Eva; Jansen, Joop H.; Dugas, Martin

    2017-01-01

    Valid variant calling results are crucial for the use of next-generation sequencing in clinical routine. However, there are numerous variant calling tools that usually differ in algorithms, filtering strategies, recommendations and thus, also in the output. We evaluated eight open-source tools regarding their ability to call single nucleotide variants and short indels with allelic frequencies as low as 1% in non-matched next-generation sequencing data: GATK HaplotypeCaller, Platypus, VarScan, LoFreq, FreeBayes, SNVer, SAMtools and VarDict. We analysed two real datasets from patients with myelodysplastic syndrome, covering 54 Illumina HiSeq samples and 111 Illumina NextSeq samples. Mutations were validated by re-sequencing on the same platform, on a different platform and expert based review. In addition we considered two simulated datasets with varying coverage and error profiles, covering 50 samples each. In all cases an identical target region consisting of 19 genes (42,322 bp) was analysed. Altogether, no tool succeeded in calling all mutations. High sensitivity was always accompanied by low precision. Influence of varying coverages- and background noise on variant calling was generally low. Taking everything into account, VarDict performed best. However, our results indicate that there is a need to improve reproducibility of the results in the context of multithreading. PMID:28233799

  20. Phenotypic diversification in vivo: Pseudomonas aeruginosa gacS- strains generate small colony variants in vivo that are distinct from in vitro variants.

    Science.gov (United States)

    Nelson, Lisa K; Stanton, M Mark; Elphinstone, Robyn E A; Helwerda, Janessa; Turner, Raymond J; Ceri, Howard

    2010-12-01

    Pseudomonas aeruginosa has long been known to produce phenotypic variants during chronic mucosal surface infections. These variants are thought to be generated to ensure bacterial survival against the diverse challenges in the mucosal environment. Studies have begun to elucidate the mechanisms by which these variants emerge in vitro; however, too little information exists on phenotypic variation in vivo to draw any links between variants generated in vitro and in vivo. Consequently, in this study, the P. aeruginosa gacS gene, which has previously been linked to the generation of small colony variants (SCVs) in vitro, was studied in an in vivo mucosal surface infection model. More specifically, the rat prostate served as a model mucosal surface to test for the appearance of SCVs in vivo following infections with P. aeruginosa gacS(-) strains. As in in vitro studies, deletion of the gacS gene led to SCV production in vivo. The appearance of these in vivo SCVs was important for the sustainability of a chronic infection. In the subset of rats in which P. aeruginosa gacS(-) did not convert to SCVs, clearance of the bacteria took place and healing of the tissue ensued. When comparing the SCVs that arose at the mucosal surface (MS-SCVs) with in vitro SCVs (IV-SCVs) from the same gacS(-) parent, some differences between the phenotypic variants were observed. Whereas both MS-SCVs and IV-SCVs formed dense biofilms, MS-SCVs exhibited a less diverse resistance profile to antimicrobial agents than IV-SCVs. Additionally, MS-SCVs were better suited to initiate an infection in the rat model than IV-SCVs. Together, these observations suggest that phenotypic variation in vivo can be important for maintenance of infection, and that in vivo variants may differ from in vitro variants generated from the same genetic parent.

  1. A rare myelin protein zero (MPZ variant alters enhancer activity in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Anthony Antonellis

    Full Text Available BACKGROUND: Myelin protein zero (MPZ is a critical structural component of myelin in the peripheral nervous system. The MPZ gene is regulated, in part, by the transcription factors SOX10 and EGR2. Mutations in MPZ, SOX10, and EGR2 have been implicated in demyelinating peripheral neuropathies, suggesting that components of this transcriptional network are candidates for harboring disease-causing mutations (or otherwise functional variants that affect MPZ expression. METHODOLOGY: We utilized a combination of multi-species sequence comparisons, transcription factor-binding site predictions, targeted human DNA re-sequencing, and in vitro and in vivo enhancer assays to study human non-coding MPZ variants. PRINCIPAL FINDINGS: Our efforts revealed a variant within the first intron of MPZ that resides within a previously described SOX10 binding site is associated with decreased enhancer activity, and alters binding of nuclear proteins. Additionally, the genomic segment harboring this variant directs tissue-relevant reporter gene expression in zebrafish. CONCLUSIONS: This is the first reported MPZ variant within a cis-acting transcriptional regulatory element. While we were unable to implicate this variant in disease onset, our data suggests that similar non-coding sequences should be screened for mutations in patients with neurological disease. Furthermore, our multi-faceted approach for examining the functional significance of non-coding variants can be readily generalized to study other loci important for myelin structure and function.

  2. Rapid Detection of Rare Deleterious Variants by Next Generation Sequencing with Optional Microarray SNP Genotype Data.

    Science.gov (United States)

    Watson, Christopher M; Crinnion, Laura A; Gurgel-Gianetti, Juliana; Harrison, Sally M; Daly, Catherine; Antanavicuite, Agne; Lascelles, Carolina; Markham, Alexander F; Pena, Sergio D J; Bonthron, David T; Carr, Ian M

    2015-09-01

    Autozygosity mapping is a powerful technique for the identification of rare, autosomal recessive, disease-causing genes. The ease with which this category of disease gene can be identified has greatly increased through the availability of genome-wide SNP genotyping microarrays and subsequently of exome sequencing. Although these methods have simplified the generation of experimental data, its analysis, particularly when disparate data types must be integrated, remains time consuming. Moreover, the huge volume of sequence variant data generated from next generation sequencing experiments opens up the possibility of using these data instead of microarray genotype data to identify disease loci. To allow these two types of data to be used in an integrated fashion, we have developed AgileVCFMapper, a program that performs both the mapping of disease loci by SNP genotyping and the analysis of potentially deleterious variants using exome sequence variant data, in a single step. This method does not require microarray SNP genotype data, although analysis with a combination of microarray and exome genotype data enables more precise delineation of disease loci, due to superior marker density and distribution.

  3. Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors

    OpenAIRE

    Jones, M.L.; Norman, J E; Morgan, N. V.; Mundell, S J; Lordkipanidze, M.; Lowe, G. C.; Daly, M E; Simpson, M.A.; Drake, S.; Watson, S P; Mumford, A D; UKGAPPS,

    2016-01-01

    Platelet responses to activating agonists are influenced by common\\ud population variants within or near G protein-coupled receptor (GPCR)\\ud genes that affect receptor activity. However, the impact of rare GPCR\\ud gene variants is unknown. We describe the rare single nucleotide variants\\ud (SNVs) in the coding and splice regions of 18 GPCR genes in\\ud 7,595 exomes from the 1,000-genomes and Exome Sequencing\\ud Project databases and in 31 cases with inherited platelet function disorders\\ud (I...

  4. Experimental verification of the identity of variant-specific surface proteins in Giardia lamblia trophozoites.

    Science.gov (United States)

    Li, Wei; Saraiya, Ashesh A; Wang, Ching C

    2013-05-21

    The cell membrane of a Giardia lamblia trophozoite is covered with a single species of variant-specific surface protein (VSP) that is replaced by another VSP every 6 to 13 generations of cell growth, possibly for an evasion of host immunity. Experimentally, only six VSP species have been verified to localize to the cell membrane thus far. By assuming that VSP contains multiple CXXC motifs, 219 vsp genes were annotated in GiardiaDB of the WB isolate. By further assuming that VSP possesses both CXXC motifs and a CRGKA tail at the C terminus, Adam et al. (BMC Genomics 11:424, 2010) identified a total of 303 potential vsp genes in Giardia WB. The discrepancies between these two assumed VSP identities have caused some confusion. Here, we used experimental approaches to further verify what is required of the structures of a VSP to localize to the surface of cell membrane. The data led to the following conclusions. (i) The C-terminal CRGKA sequence is not essential for localizing VSPs to the cell membrane. (ii) A "motif 1" of 45 residues, consisting of two CXXCs separated by 12 to 15 amino acid residues, located close to the C terminus and a hydrophobic "motif 2" of 38 residues at the C terminus are both essential and sufficient for localizing the protein to the cell membrane. (ii) An N-terminal sequence upstream from motif 1 is not required for targeting VSPs to the cell membrane. By these criteria, we are able to identify 73 open reading frames as the putative vsp genes in Giardia. IMPORTANCE The intestinal pathogen Giardia lamblia expresses only one variant-specific surface protein (VSP) on the cell membrane surface at a given time, but it changes spontaneously every 6 to 13 generations of growth, presumably for evading the host immunity. Only 6 VSPs have been empirically shown to localize to the cell membrane surface thus far. Here, we used mutations of VSPs and methods of identifying their locations in Giardia cells and found that a "motif 1" of 45 residues

  5. Characterization of murine SIRT3 transcript variants and corresponding protein products

    Science.gov (United States)

    SIRT3 is one of the seven mammalian sirtuin homologs of the yeast SIR2 gene. SIRT3 possesses NAD(+)-dependent protein deacetylase activity. Recent studies indicate that the murine SIRT3 gene expresses different transcript variants, resulting in three possible SIRT3 protein isoforms with various leng...

  6. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Sternberg, Claus; Poulsen, Lars K.

    1998-01-01

    Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria ia is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been con...

  7. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Sternberg, Claus; Poulsen, Lars K.

    1998-01-01

    Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria ia is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been...

  8. Protein conformational perturbations in hereditary amyloidosis: Differential impact of single point mutations in ApoAI amyloidogenic variants.

    Science.gov (United States)

    Del Giudice, Rita; Arciello, Angela; Itri, Francesco; Merlino, Antonello; Monti, Maria; Buonanno, Martina; Penco, Amanda; Canetti, Diana; Petruk, Ganna; Monti, Simona Maria; Relini, Annalisa; Pucci, Piero; Piccoli, Renata; Monti, Daria Maria

    2016-02-01

    Amyloidoses are devastating diseases characterized by accumulation of misfolded proteins which aggregate in fibrils. Specific gene mutations in Apolipoprotein A I (ApoAI) are associated with systemic amyloidoses. Little is known on the effect of mutations on ApoAI structure and amyloid properties. Here we performed a physico-chemical characterization of L75P- and L174S-amyloidogenic ApoAI (AApoAI) variants to shed light on the effects of two single point mutations on protein stability, proteolytic susceptibility and aggregation propensity. Both variants are destabilized in their N-terminal region and generate fibrils with different morphological features. L75P-AApoAI is significantly altered in its conformation and compactness, whereas a more flexible and pronounced aggregation-competent state is associated to L174S-AApoAI. These observations point out how single point mutations in ApoAI gene evocate differences in the physico-chemical and conformational behavior of the corresponding protein variants, with the common feature of diverting ApoAI from its natural role towards a pathogenic pathway.

  9. Structure-function studies of an engineered scaffold protein derived from stefin A. I: Development of the SQM variant.

    Science.gov (United States)

    Hoffmann, Toni; Stadler, Lukas Kurt Josef; Busby, Michael; Song, Qifeng; Buxton, Anthony T; Wagner, Simon D; Davis, Jason J; Ko Ferrigno, Paul

    2010-05-01

    Non-antibody scaffold proteins are used for a range of applications, especially the assessment of protein-protein interactions within human cells. The search for a versatile, robust and biologically neutral scaffold previously led us to design STM (stefin A triple mutant), a scaffold derived from the intracellular protease inhibitor stefin A. Here, we describe five new STM-based scaffold proteins that contain modifications designed to further improve the versatility of our scaffold. In a step-by-step approach, we introduced restriction sites in the STM open reading frame that generated new peptide insertion sites in loop 1, loop 2 and the N-terminus of the scaffold protein. A second restriction site in 'loop 2' allows substitution of the native loop 2 sequence with alternative oligopeptides. None of the amino acid changes interfered significantly with the folding of the STM variants as assessed by circular dichroism spectroscopy. Of the five scaffold variants tested, one (stefin A quadruple mutant, SQM) was chosen as a versatile, stable scaffold. The insertion of epitope tags at varying positions showed that inserts into loop 1, attempted here for the first time, were generally well tolerated. However, N-terminal insertions of epitope tags in SQM had a detrimental effect on protein expression.

  10. Correlation of rare coding variants in the gene encoding human glucokinase regulatory protein with phenotypic, cellular, and kinetic outcomes.

    Science.gov (United States)

    Rees, Matthew G; Ng, David; Ruppert, Sarah; Turner, Clesson; Beer, Nicola L; Swift, Amy J; Morken, Mario A; Below, Jennifer E; Blech, Ilana; Mullikin, James C; McCarthy, Mark I; Biesecker, Leslie G; Gloyn, Anna L; Collins, Francis S

    2012-01-01

    Defining the genetic contribution of rare variants to common diseases is a major basic and clinical science challenge that could offer new insights into disease etiology and provide potential for directed gene- and pathway-based prevention and treatment. Common and rare nonsynonymous variants in the GCKR gene are associated with alterations in metabolic traits, most notably serum triglyceride levels. GCKR encodes glucokinase regulatory protein (GKRP), a predominantly nuclear protein that inhibits hepatic glucokinase (GCK) and plays a critical role in glucose homeostasis. The mode of action of rare GCKR variants remains unexplored. We identified 19 nonsynonymous GCKR variants among 800 individuals from the ClinSeq medical sequencing project. Excluding the previously described common missense variant p.Pro446Leu, all variants were rare in the cohort. Accordingly, we functionally characterized all variants to evaluate their potential phenotypic effects. Defects were observed for the majority of the rare variants after assessment of cellular localization, ability to interact with GCK, and kinetic activity of the encoded proteins. Comparing the individuals with functional rare variants to those without such variants showed associations with lipid phenotypes. Our findings suggest that, while nonsynonymous GCKR variants, excluding p.Pro446Leu, are rare in individuals of mixed European descent, the majority do affect protein function. In sum, this study utilizes computational, cell biological, and biochemical methods to present a model for interpreting the clinical significance of rare genetic variants in common disease.

  11. A 17.6 kbp region located upstream of the rabbit WAP gene directs high level expression of a functional human protein variant in transgenic mouse milk

    NARCIS (Netherlands)

    Bischoff, Rainer; Degryse, E.; Perraud, F.; Dalemans, W.; Ali-Hadji, D.; Thepot, D.; Devinoy, E.; Houdebine, L.M.; Pavirani, A.

    1992-01-01

    We have investigated whether DNA regions present in the rabbit whey acidic protein (WAP) promoter/5' flanking sequence could potentially confer, in vivo, high level expression of reporter genes. Transgenic mice were generated expressing a variant of human alpha 1-antitrypsin, which has inhibitory ac

  12. Diversity and Impact of Rare Variants in Genes Encoding the Platelet G Protein-Coupled Receptors

    Science.gov (United States)

    Jones, Matthew L.; Norman, Jane E.; Morgan, Neil V.; Mundell, Stuart J.; Lordkipanidzé, Marie; Lowe, Gillian C.; Daly, Martina E.; Simpson, Michael A.; Drake, Sian; Watson, Steve P.

    2015-01-01

    Summary Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70% had global minor allele frequency (MAF) low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes. PMID:25567036

  13. Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors.

    Science.gov (United States)

    Jones, Matthew L; Norman, Jane E; Morgan, Neil V; Mundell, Stuart J; Lordkipanidzé, Marie; Lowe, Gillian C; Daly, Martina E; Simpson, Michael A; Drake, Sian; Watson, Steve P; Mumford, Andrew D

    2015-04-01

    Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70 % had global minor allele frequency (MAF) low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.

  14. Impact of predicted protein-truncating genetic variants on the human transcriptome

    Science.gov (United States)

    Rivas, Manuel A.; Pirinen, Matti; Conrad, Donald F.; Lek, Monkol; Tsang, Emily K.; Karczewski, Konrad J.; Maller, Julian B.; Kukurba, Kimberly R.; DeLuca, David; Fromer, Menachem; Ferreira, Pedro G.; Smith, Kevin S.; Zhang, Rui; Zhao, Fengmei; Banks, Eric; Poplin, Ryan; Ruderfer, Douglas; Purcell, Shaun M.; Tukiainen, Taru; Minikel, Eric V.; Stenson, Peter D.; Cooper, David N.; Huang, Katharine H.; Sullivan, Timothy J.; Nedzel, Jared; Bustamante, Carlos D.; Li, Jin Billy; Daly, Mark J.; Guigo, Roderic; Donnelly, Peter; Ardlie, Kristin; Sammeth, Michael; Dermitzakis, Emmanouil; McCarthy, Mark I.; Montgomery, Stephen B.; Lappalainen, Tuuli; MacArthur, Daniel G.

    2015-01-01

    Accurate prediction of the functional impact of genetic variation is critical for clinical genome interpretation. We systematically characterized the transcriptome effects of protein-truncating variants (PTVs), a class of variants expected to have profound impacts on gene function, using data from the Genotype-Tissue Expression (GTEx) and Geuvadis projects. We quantitate tissue-specific and positional effects on nonsense-mediated transcript decay, and present an improved predictive model for this decay. We directly measure the impact of variants both proximal and distal to splice junctions. Furthermore, we find that robustness to heterozygous gene inactivation is not due to dosage compensation. Our results illustrate the value of transcriptome data in the functional interpretation of genetic variants. PMID:25954003

  15. Recombinational joints in a simian virus 40 variant generated in a persistent infection.

    Science.gov (United States)

    Norkin, L C; Piatak, M

    1982-12-01

    SP1, a viable simian virus 40 (SV40) variant isolated from a persistent infection of rhesus monkey kidney cells, contains sequence rearrangements in the untranslated region of the SV40 genome which are transcribed into late mRNA leader sequences and in the region which encodes the large T antigen. Nucleotide sequences about the recombinational junctions in SP1 were determined. The sequence data show that in most instances there was not extensive homology between recombining sequences. The recombinant sequences are discussed with respect to the mechanisms by which they might have been generated.

  16. Technical and Economic Advantages of Turbo-Drive Variant with TRB Turbines over Turbo-Generator Variant in Low Power Engineering

    OpenAIRE

    N. V. Panteley

    2008-01-01

    The paper contains a comparative analysis of turbo-driven variant application with TRB turbines and turbo-generator variant in the low power engineering. High efficiency of steam-turbine drive application with TRB turbines is proved by calculation which is made on the basis of a SE-1250-140 network pump at one of the boilers of the Republic of Belarus taken as an example. Calculation has been made for the following steam parameters: turbo-drive input – 12 kgs/cm and 250 and parameters behind ...

  17. Detection of type 1 prion protein in variant Creutzfeldt-Jakob disease

    NARCIS (Netherlands)

    Yull, H.M.; Ritchie, D.L.; Langeveld, J.P.M.; Zijderveld, van F.G.; Bruce, M.E.; Ironside, J.W.; Head, M.W.

    2006-01-01

    Molecular typing of the abnormal form of the prion protein (PrPSc) has come to be regarded as a powerful tool in the investigation of the prion diseases. All evidence thus far presented indicates a single PrPSc molecular type in variant Creutzfeldt-Jakob disease (termed type 2B), presumably resultin

  18. Functional assessment of population and tumor-associated APE1 protein variants.

    Directory of Open Access Journals (Sweden)

    Jennifer L Illuzzi

    Full Text Available Apurinic/apyrimidinic endonuclease 1 (APE1 is the predominant AP site repair enzyme in mammals. APE1 also maintains 3'-5' exonuclease and 3'-repair activities, and regulates transcription factor DNA binding through its REF-1 function. Since complete or severe APE1 deficiency leads to embryonic lethality and cell death, it has been hypothesized that APE1 protein variants with slightly impaired function will contribute to disease etiology. Our data indicate that except for the endometrial cancer-associated APE1 variant R237C, the polymorphic variants Q51H, I64V and D148E, the rare population variants G241R, P311S and A317V, and the tumor-associated variant P112L exhibit normal thermodynamic stability of protein folding; abasic endonuclease, 3'-5' exonuclease and REF-1 activities; coordination during the early steps of base excision repair; and intracellular distribution when expressed exogenously in HeLa cells. The R237C mutant displayed reduced AP-DNA complex stability, 3'-5' exonuclease activity and 3'-damage processing. Re-sequencing of the exonic regions of APE1 uncovered no novel amino acid substitutions in the 60 cancer cell lines of the NCI-60 panel, or in HeLa or T98G cancer cell lines; only the common D148E and Q51H variants were observed. Our results indicate that APE1 missense mutations are seemingly rare and that the cancer-associated R237C variant may represent a reduced-function susceptibility allele.

  19. The Clinical Next-Generation Sequencing Database: A Tool for the Unified Management of Clinical Information and Genetic Variants to Accelerate Variant Pathogenicity Classification.

    Science.gov (United States)

    Nishio, Shin-Ya; Usami, Shin-Ichi

    2017-03-01

    Recent advances in next-generation sequencing (NGS) have given rise to new challenges due to the difficulties in variant pathogenicity interpretation and large dataset management, including many kinds of public population databases as well as public or commercial disease-specific databases. Here, we report a new database development tool, named the "Clinical NGS Database," for improving clinical NGS workflow through the unified management of variant information and clinical information. This database software offers a two-feature approach to variant pathogenicity classification. The first of these approaches is a phenotype similarity-based approach. This database allows the easy comparison of the detailed phenotype of each patient with the average phenotype of the same gene mutation at the variant or gene level. It is also possible to browse patients with the same gene mutation quickly. The other approach is a statistical approach to variant pathogenicity classification based on the use of the odds ratio for comparisons between the case and the control for each inheritance mode (families with apparently autosomal dominant inheritance vs. control, and families with apparently autosomal recessive inheritance vs. control). A number of case studies are also presented to illustrate the utility of this database.

  20. The Quest for Rare Variants: Pooled Multiplexed Next Generation Sequencing in Plants

    Directory of Open Access Journals (Sweden)

    Fabio eMarroni

    2012-06-01

    Full Text Available Next generation sequencing (NGS instruments produce an unprecedented amount of sequence data at contained costs. This gives researchers the possibility of designing studies with adequate power to identify rare variants at a fraction of the economic and labor resources required by individual Sanger sequencing. As of today, only three research groups working in plant sciences have exploited this potentiality. They showed that pooled NGS can provide results in excellent agreement with those obtained by individual Sanger sequencing. Aim of this review is to convey to the reader the general ideas underlying the use of pooled NGS for the identification of rare variants. To facilitate a thorough understanding of the possibilities of the method we will explain in detail the variations in study design and discuss their advantages and disadvantages. We will show that information on allele frequency obtained by pooled next generation sequencing can be used to accurately compute basic population genetics indexes such as allele frequency, nucleotide diversity and Tajima’s D. Finally we will discuss applications and future perspectives of the multiplexed NGS approach.

  1. Human activated protein C variants in a rat model of arterial thrombosis

    Directory of Open Access Journals (Sweden)

    Dahlbäck Björn

    2008-10-01

    Full Text Available Abstract Background Activated protein C (APC inhibits coagulation by degrading activated factor V (FVa and factor VIII (FVIIIa, protein S (PS functioning as a cofactor to APC. Methods By mutagenesis of the vitamin K-dependent Gla domain of APC, we have recently created an APC variant having enhanced anticoagulant activity due to increased affinity for negatively charged phospholipid membranes. In the present study, the potential antithrombotic effects of this APC variant, and of a variant APC that is additionally mutated in the serine protease domain, have been evaluated in a blind randomized study in a rat model of arterial thrombosis. In this model, we have previously found the combination of bovine APC and PS to be highly antithrombotic. Four treatment groups each containing 10 rats were, in a blind random fashion, given intravenous bolus injections of wild-type or mutant variants of APC (0.8 mg/kg together with human PS (0.6 mg/kg or human PS (0.6 mg/kg alone. A control group with 20 animals where given vehicle only. Results A trend to increased patency rates was noted in a group receiving one of the APC variants, but it did not reach statistical significance. Conclusion In conclusion, administration of human APC variants having enhanced anticoagulant efficacy together with human PS in a rat model of arterial thrombosis did not give an efficient antithrombotic effect. The lack of effect may be due to species-specific differences between the human protein C system and the rat hemostatic system.

  2. Generation of red fluorescent protein transgenic dogs.

    Science.gov (United States)

    Hong, So Gun; Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Park, Jung Eun; Kang, Jung Taek; Koo, Ok Jae; Kim, Teoan; Kwon, Mo Sun; Koo, Bon Chul; Ra, Jeong Chan; Kim, Dae Yong; Ko, CheMyong; Lee, Byeong Chun

    2009-05-01

    Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene-expressing construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP-fibroblasts. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP.

  3. Next-Generation Sequencing for Binary Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Bernhard eSuter

    2015-12-01

    Full Text Available The yeast two-hybrid (Y2H system exploits host cell genetics in order to display binary protein-protein interactions (PPIs via defined and selectable phenotypes. Numerous improvements have been made to this method, adapting the screening principle for diverse applications, including drug discovery and the scale-up for proteome wide interaction screens in human and other organisms. Here we discuss a systematic workflow and analysis scheme for screening data generated by Y2H and related assays that includes high-throughput selection procedures, readout of comprehensive results via next-generation sequencing (NGS, and the interpretation of interaction data via quantitative statistics. The novel assays and tools will serve the broader scientific community to harness the power of NGS technology to address PPI networks in health and disease. We discuss examples of how this next-generation platform can be applied to address specific questions in diverse fields of biology and medicine.

  4. A hetero-micro-seeding strategy for readily crystallizing closely related protein variants.

    Science.gov (United States)

    Islam, Mohammad M; Kuroda, Yutaka

    2017-09-01

    Protein crystallization remains difficult to rationalize and screening for optimal crystallization conditions is a tedious and time consuming procedure. Here, we report a hetero-micro-seeding strategy for producing high resolution crystals of closely related protein variants, where micro crystals from a readily crystallized variant are used as seeds to develop crystals of other variants less amenable to crystallization. We applied this strategy to Bovine Pancreatic Trypsin Inhibitor (BPTI) variants, which would not crystallize using standard crystallization practice. Out of six variants in our analysis, only one called BPTI-[5,55]A14G formed well behaving crystals; and the remaining five (A14GA38G, A14GA38V, A14GA38L, A14GA38I, and A14GA38K) could be crystallized only using micro-seeds from the BPTI-[5,55]A14G crystal. All hetero-seeded crystals diffracted at high resolution with minimum mosaicity, retaining the same space group and cell dimension. Moreover, hetero-micro-seeding did not introduce any biases into the mutant's structure toward the seed structure, as demonstrated by A14GA38I structures solved using micro-seeds from A14GA38G, A14GA38L and A14GA38I. Though hetero-micro-seeding is a simple and almost naïve strategy, this is the first direct demonstration of its workability. We believe that hetero-micro-seeding, which is contrasting with the popular idea that crystallization requires highly purified proteins, could contribute a new tool for rapidly solving protein structures in mutational analysis studies. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues

    Science.gov (United States)

    Suzuki, Takashi; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5′-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5′-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity. PMID:27256115

  6. Origin of carrier generation in photovoltaic perovskite variant Cs2SnI6

    CERN Document Server

    Xiao, Zewen; Kamiya, Toshio

    2015-01-01

    Cs2SnI6 is an air-stable & non-toxic variant of perovskite-type photovoltaic materials. In this letter, stability of intrinsic defects in Cs2SnI6 was examined by density functional theory calculations. We found that iodine vacancy and tin interstitial are the dominant defects, mainly responsible for the intrinsic n-type conductivity in Cs2SnI6. However, the transition levels of the dominant defects are deep, which makes it difficult to achieve high-density n-type doping. Tin vacancy is expected for p-type doping, but it has a very high formation energy > 3.6 eV because of the strong Sn-I covalent bonds and can hardly be generated. Instead, cesium vacancy is formed at an extremely Cs-poor condition and explains already-reported p-type conductivity by SnI2 doping.

  7. Evaluating rare variants in complex disorders using next-generation sequencing.

    Science.gov (United States)

    Ezewudo, Matthew; Zwick, Michael E

    2013-04-01

    Determining the genetic architecture of liability for complex neuropsychiatric disorders like autism spectrum disorders and schizophrenia poses a tremendous challenge for contemporary biomedical research. Here we discuss how genetic studies first tested, and rejected, the hypothesis that common variants with large effects account for the prevalence of these disorders. We then explore how the discovery of structural variation has contributed to our understanding of the etiology of these disorders. The rise of fast and inexpensive oligonucleotide sequencing and methods of targeted enrichment and their influence on the search for rare genetic variation contributing to complex neuropsychiatric disorders is the next focus of our article. Finally, we consider the technical challenges and future prospects for the use of next-generation sequencing to reveal the complex genetic architecture of complex neuropsychiatric disorders in both research and the clinical settings.

  8. Alternative splice variants in TIM barrel proteins from human genome correlate with the structural and evolutionary modularity of this versatile protein fold.

    Science.gov (United States)

    Ochoa-Leyva, Adrián; Montero-Morán, Gabriela; Saab-Rincón, Gloria; Brieba, Luis G; Soberón, Xavier

    2013-01-01

    After the surprisingly low number of genes identified in the human genome, alternative splicing emerged as a major mechanism to generate protein diversity in higher eukaryotes. However, it is still not known if its prevalence along the genome evolution has contributed to the overall functional protein diversity or if it simply reflects splicing noise. The (βα)8 barrel or TIM barrel is one of the most frequent, versatile, and ancient fold encountered among enzymes. Here, we analyze the structural modifications present in TIM barrel proteins from the human genome product of alternative splicing events. We found that 87% of all splicing events involved deletions; most of these events resulted in protein fragments that corresponded to the (βα)2, (βα)4, (βα)5, (βα)6, and (βα)7 subdomains of TIM barrels. Because approximately 7% of all the splicing events involved internal β-strand substitutions, we decided, based on the genomic data, to design β-strand and α-helix substitutions in a well-studied TIM barrel enzyme. The biochemical characterization of one of the chimeric variants suggests that some of the splice variants in the human genome with β-strand substitutions may be evolving novel functions via either the oligomeric state or substrate specificity. We provide results of how the splice variants represent subdomains that correlate with the independently folding and evolving structural units previously reported. This work is the first to observe a link between the structural features of the barrel and a recurrent genetic mechanism. Our results suggest that it is reasonable to expect that a sizeable fraction of splice variants found in the human genome represent structurally viable functional proteins. Our data provide additional support for the hypothesis of the origin of the TIM barrel fold through the assembly of smaller subdomains. We suggest a model of how nature explores new proteins through alternative splicing as a mechanism to diversify the

  9. Alternative splice variants in TIM barrel proteins from human genome correlate with the structural and evolutionary modularity of this versatile protein fold.

    Directory of Open Access Journals (Sweden)

    Adrián Ochoa-Leyva

    Full Text Available After the surprisingly low number of genes identified in the human genome, alternative splicing emerged as a major mechanism to generate protein diversity in higher eukaryotes. However, it is still not known if its prevalence along the genome evolution has contributed to the overall functional protein diversity or if it simply reflects splicing noise. The (βα8 barrel or TIM barrel is one of the most frequent, versatile, and ancient fold encountered among enzymes. Here, we analyze the structural modifications present in TIM barrel proteins from the human genome product of alternative splicing events. We found that 87% of all splicing events involved deletions; most of these events resulted in protein fragments that corresponded to the (βα2, (βα4, (βα5, (βα6, and (βα7 subdomains of TIM barrels. Because approximately 7% of all the splicing events involved internal β-strand substitutions, we decided, based on the genomic data, to design β-strand and α-helix substitutions in a well-studied TIM barrel enzyme. The biochemical characterization of one of the chimeric variants suggests that some of the splice variants in the human genome with β-strand substitutions may be evolving novel functions via either the oligomeric state or substrate specificity. We provide results of how the splice variants represent subdomains that correlate with the independently folding and evolving structural units previously reported. This work is the first to observe a link between the structural features of the barrel and a recurrent genetic mechanism. Our results suggest that it is reasonable to expect that a sizeable fraction of splice variants found in the human genome represent structurally viable functional proteins. Our data provide additional support for the hypothesis of the origin of the TIM barrel fold through the assembly of smaller subdomains. We suggest a model of how nature explores new proteins through alternative splicing as a mechanism to

  10. Morphology and protein molecular weight analysis from three variant of oil palm pollen; Dura, Pisifera, and Tenera

    Science.gov (United States)

    Priambodo, R.; Witarto, A. B.; Salamah, A.; Setiorini, Triyono, D.; Bowolaksono, A.

    2017-07-01

    Oil palm is a plant that widely cultivated in Indonesia, with an area of about 11 million hectares in 2014. There are three main variants that most cultivated; Dura, Pisifera, and Tenera. Oil palm pollen was spread through the wind. The very wide area of oil palm plantation and those characteristics of oil palm pollen dispersion makes oil palm pollen may give negative effect to the people around plantation, such as an allergy. The research on the morphology and protein characters of the oil palm pollen from three variants has not done yet. This research aims to observe the morphology and protein character from three variants of oil palm pollen. The study begins with the pollen collection from three variants of oil palm. Oil palm pollen was observed using the light and scanning electron microscope. Oil palm pollen protein was extracted and the molecular weight of these proteins was analyzed. The result of this research was the morphology character from three variants of oil palm pollen have successfully been observed. Those three variant of oil palm have no differences structures; triangular shaped with round edge, tricolpate with connected colpus aperture, psilate exine ornamentation at the front side and peripheral side, while at the back side has microreticulate exine ornamentation. Three variants of oil palm pollen protein show the same characteristics. The molecular weight of the protein was ranged from 10-00 KDa. The information can be useful for the next research to figure out component of proteins inside the oil palm pollen.

  11. Multiple Gene Variants in Hypertrophic Cardiomyopathy in the Era of Next-Generation Sequencing.

    Science.gov (United States)

    Burns, Charlotte; Bagnall, Richard D; Lam, Lien; Semsarian, Christopher; Ingles, Jodie

    2017-08-01

    Multiple likely pathogenic/pathogenic (LP/P; ≥2) variants in patients with hypertrophic cardiomyopathy were described 10 years ago with a prevalence of 5%. We sought to re-examine the significance of multiple rare variants in patients with hypertrophic cardiomyopathy in the setting of comprehensive and targeted panels. Of 758 hypertrophic cardiomyopathy probands, we included 382 with ≥45 cardiomyopathy genes screened. There were 224 (59%) with ≥1 rare variant (allele frequency ≤0.02%). Variants were analyzed using varying sized gene panels to represent comprehensive or targeted testing. Based on a 45-gene panel, 127 (33%) had a LP/P variant, 139 (36%) had variants of uncertain significance, and 66 (17%) had multiple rare variants. A targeted 8-gene panel yielded 125 (32%) LP/P variants, 52 (14%) variants of uncertain significance, and 14 (4%) had multiple rare variants. No proband had 2 LP/P variants. Including affected family members (total n=412), cluster-adjusted analyses identified a phenotype effect, with younger age (odds ratio, 0.95; 95% confidence interval, 0.92-0.98; P=0.004) and family history of sudden cardiac death (odds ratio, 3.5; 95% confidence interval, 1.3-9.9; P=0.02) significantly more likely in multiple versus single variant patients when considering an 8-gene panel but not larger panels. Those with multiple variants had worse event-free survival from all-cause death, cardiac transplantation, and cardiac arrest (log-rank P=0.008). No proband had multiple LP/P variants in contrast to previous reports. However, multiple rare variants regardless of classification were seen in 4% and contributed to earlier disease onset and cardiac events. Our findings support a cumulative variant hypothesis in hypertrophic cardiomyopathy. © 2017 American Heart Association, Inc.

  12. Lin28 induces resistance to anti-androgens via promotion of AR splice variant generation.

    Science.gov (United States)

    Tummala, Ramakumar; Nadiminty, Nagalakshmi; Lou, Wei; Evans, Christopher P; Gao, Allen C

    2016-04-01

    Prostate cancer (PCa) is androgen-dependent initially and progresses to a castration-resistant state after androgen deprivation therapy. Treatment options for castration-resistant PCa include the potent second-generation anti-androgen enzalutamide or CYP17A1 inhibitor abiraterone. Recent clinical observations point to the development of resistance to these therapies which may be mediated by constitutively active alternative splice variants of the androgen receptor (AR). Sensitivity of LNCaP cells overexpressing Lin28 (LN-Lin28) to enzalutamide, abiraterone, or bicalutamide was compared to that of control LN-neo cells using cell growth assays, proliferation assays using MTT, anchorage-dependent clonogenic ability assays and soft agar assays. Ability of LN-Lin28 cells to maintain AR activation after treatment with enzalutamide, abiraterone, or bicalutamide was tested using immunofluorescence, Western blotting, ChIP assays, and qRT-PCR. Importance of Lin28 in enzalutamide resistance was assessed by the downregulation of Lin28 expression in C4-2B and 22Rv1 cells chronically treated with enzalutamide. Requirement for sustained AR signaling in LN-Lin28 cells was examined by the downregulation of either full length AR or AR-V7 using siRNA. We show that Lin28 promotes the development of resistance to currently used targeted therapeutics by enhancing the expression of AR splice variants such as AR-V7. PCa cells overexpressing Lin28 exhibit resistance to treatment with enzalutamide, abiraterone, or bicalutamide. Downregulation of Lin28 resensitizes enzalutamide-resistant PCa cells to enzalutamide treatment. We also show that the upregulation of splicing factors such as hnRNPA1 by Lin28 may mediate the enhanced generation of AR splice variants in Lin28-expressing cells. Our findings suggest that Lin28 plays a key role in the acquisition of resistance to AR-targeted therapies by PCa cells and establish the importance of Lin28 in PCa progression. © 2015 Wiley Periodicals, Inc.

  13. Learning generative models for protein fold families.

    Science.gov (United States)

    Balakrishnan, Sivaraman; Kamisetty, Hetunandan; Carbonell, Jaime G; Lee, Su-In; Langmead, Christopher James

    2011-04-01

    We introduce a new approach to learning statistical models from multiple sequence alignments (MSA) of proteins. Our method, called GREMLIN (Generative REgularized ModeLs of proteINs), learns an undirected probabilistic graphical model of the amino acid composition within the MSA. The resulting model encodes both the position-specific conservation statistics and the correlated mutation statistics between sequential and long-range pairs of residues. Existing techniques for learning graphical models from MSA either make strong, and often inappropriate assumptions about the conditional independencies within the MSA (e.g., Hidden Markov Models), or else use suboptimal algorithms to learn the parameters of the model. In contrast, GREMLIN makes no a priori assumptions about the conditional independencies within the MSA. We formulate and solve a convex optimization problem, thus guaranteeing that we find a globally optimal model at convergence. The resulting model is also generative, allowing for the design of new protein sequences that have the same statistical properties as those in the MSA. We perform a detailed analysis of covariation statistics on the extensively studied WW and PDZ domains and show that our method out-performs an existing algorithm for learning undirected probabilistic graphical models from MSA. We then apply our approach to 71 additional families from the PFAM database and demonstrate that the resulting models significantly out-perform Hidden Markov Models in terms of predictive accuracy.

  14. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

    Energy Technology Data Exchange (ETDEWEB)

    Xi, T; Jones, I M; Mohrenweiser, H W

    2003-11-03

    Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of the variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

  15. wKinMut-2: Identification and Interpretation of Pathogenic Variants in Human Protein Kinases

    DEFF Research Database (Denmark)

    Vazquez, Miguel; Pons, Tirso; Brunak, Søren;

    2016-01-01

    is often scattered across different sources, which makes the integrative analysis complex and laborious. wKinMut-2 constitutes a solution to facilitate the interpretation of the consequences of human protein kinase variation. Nine methods predict their pathogenicity, including a kinase-specific random...... forest approach. To understand the biological mechanisms causative of human diseases and cancer, information from pertinent reference knowledgebases and the literature is automatically mined, digested and homogenized. Variants are visualized in their structural contexts and residues affecting catalytic...

  16. wKinMut-2: Identification and Interpretation of Pathogenic Variants in Human Protein Kinases

    DEFF Research Database (Denmark)

    Vazquez, Miguel; Pons, Tirso; Brunak, Søren

    2016-01-01

    is often scattered across different sources, which makes the integrative analysis complex and laborious. wKinMut-2 constitutes a solution to facilitate the interpretation of the consequences of human protein kinase variation. Nine methods predict their pathogenicity, including a kinase-specific random...... forest approach. To understand the biological mechanisms causative of human diseases and cancer, information from pertinent reference knowledgebases and the literature is automatically mined, digested and homogenized. Variants are visualized in their structural contexts and residues affecting catalytic...

  17. A naturally occurring variant of the human prion protein completely prevents prion disease.

    Science.gov (United States)

    Asante, Emmanuel A; Smidak, Michelle; Grimshaw, Andrew; Houghton, Richard; Tomlinson, Andrew; Jeelani, Asif; Jakubcova, Tatiana; Hamdan, Shyma; Richard-Londt, Angela; Linehan, Jacqueline M; Brandner, Sebastian; Alpers, Michael; Whitfield, Jerome; Mead, Simon; Wadsworth, Jonathan D F; Collinge, John

    2015-06-25

    Mammalian prions, transmissible agents causing lethal neurodegenerative diseases, are composed of assemblies of misfolded cellular prion protein (PrP). A novel PrP variant, G127V, was under positive evolutionary selection during the epidemic of kuru--an acquired prion disease epidemic of the Fore population in Papua New Guinea--and appeared to provide strong protection against disease in the heterozygous state. Here we have investigated the protective role of this variant and its interaction with the common, worldwide M129V PrP polymorphism. V127 was seen exclusively on a M129 PRNP allele. We demonstrate that transgenic mice expressing both variant and wild-type human PrP are completely resistant to both kuru and classical Creutzfeldt-Jakob disease (CJD) prions (which are closely similar) but can be infected with variant CJD prions, a human prion strain resulting from exposure to bovine spongiform encephalopathy prions to which the Fore were not exposed. Notably, mice expressing only PrP V127 were completely resistant to all prion strains, demonstrating a different molecular mechanism to M129V, which provides its relative protection against classical CJD and kuru in the heterozygous state. Indeed, this single amino acid substitution (G→V) at a residue invariant in vertebrate evolution is as protective as deletion of the protein. Further study in transgenic mice expressing different ratios of variant and wild-type PrP indicates that not only is PrP V127 completely refractory to prion conversion but acts as a potent dose-dependent inhibitor of wild-type prion propagation.

  18. Identification of Novel Milk Protein Gene Variants in Sahiwal Cattle Breed of Pakistan

    Directory of Open Access Journals (Sweden)

    Shahlla N. Mir

    2013-02-01

    Full Text Available This novel study was aimed at identification of new genetic variants in Sahiwal cattle breed of Pakistan and determined the effects of these variants on milk yield. Five major milk protein genes in Sahiwal cattle were analyzed and two single nucleotide polymorphisms identified through bi-directional sequencing. These include A to T in exon XI at position 11462 of the alpha s1 casein gene; resulting in a Glutamic Acid (GAA to Aspartic acid (GAU substitution at position 84 of alpha s1 casein protein and T to C change at position 8491 of the exon VII in beta-casein gene resulting in a Valine to Alanine substitution at position 197 of beta casein protein. Amplification Refractory Mutation System (ARMS and SNaPshot genotyping protocols were optimized for genotyping new genetic variants. The genotypes in both the alpha-s1 casein and beta casein genes were found associated with milk yield but their influence was not statistically significant. However, the least square means of milk yield for TT genotypes of alpha s1 casein and of beta casein genes were higher compared to other genotypes.

  19. Monoclonal antibody-escape variant of dengue virus serotype 1: Genetic composition and envelope protein expression.

    Science.gov (United States)

    Chem, Y K; Chua, K B; Malik, Y; Voon, K

    2015-06-01

    Monoclonal antibody-escape variant of dengue virus type 1 (MabEV DEN-1) was discovered and isolated in an outbreak of dengue in Klang Valley, Malaysia from December 2004 to March 2005. This study was done to investigate whether DEN152 (an isolate of MabEV DEN-1) is a product of recombination event or not. In addition, the non-synonymous mutations that correlate with the monoclonal antibody-escape variant were determined in this study. The genomes of DEN152 and two new DEN-1 isolates, DENB04 and DENK154 were completely sequenced, aligned, and compared. Phylogenetic tree was plotted and the recombination event on DEN152 was investigated. DEN152 is sub-grouped under genotype I and is closely related genetically to a DEN-1 isolated in Japan in 2004. DEN152 is not a recombinant product of any parental strains. Four amino acid substitutions were unique only to DEN 152. These amino acid substitutions were (Ser)[326](Leu), (Ser)[340](Leu) at the deduced E protein, (Ile)[250](Thr) at NS1 protein, and (Thr)[41](Ser) at NS5 protein. Thus, DEN152 is an isolate of the emerging monoclonal antibody-escape variant DEN-1 that escaped diagnostic laboratory detection.

  20. Technical and Economic Advantages of Turbo-Drive Variant with TRB Turbines over Turbo-Generator Variant in Low Power Engineering

    Directory of Open Access Journals (Sweden)

    N. V. Panteley

    2008-01-01

    Full Text Available The paper contains a comparative analysis of turbo-driven variant application with TRB turbines and turbo-generator variant in the low power engineering. High efficiency of steam-turbine drive application with TRB turbines is proved by calculation which is made on the basis of a SE-1250-140 network pump at one of the boilers of the Republic of Belarus taken as an example. Calculation has been made for the following steam parameters: turbo-drive input – 12 kgs/cm and 250 and parameters behind turbo-drive – 1kgs/cm. Comparative analysis has been prepared on the basis of data presented by BelNIPIenergoprom in respect of regional boiler No.3 of Mogilev.

  1. Handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins in transgenic mice

    DEFF Research Database (Denmark)

    Kragh, Peter M; Pedersen, Christina B; Schmidt, Stine P;

    2007-01-01

    Abstract To investigate the in vivo handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins, three transgenic mouse lines were produced by pronuclear injection of cDNA encoding the wild-type, hSCAD-wt, and two disease causing folding variants hSCAD-319C > T and hSCAD-625G > A...

  2. Influence of HFE variants and cellular iron on monocyte chemoattractant protein-1

    Directory of Open Access Journals (Sweden)

    Simmons Zachary

    2009-02-01

    Full Text Available Abstract Background Polymorphisms in the MHC class 1-like gene known as HFE have been proposed as genetic modifiers of neurodegenerative diseases that include neuroinflammation as part of the disease process. Variants of HFE are relatively common in the general population and are most commonly associated with iron overload, but can promote subclinical cellular iron loading even in the absence of clinically identified disease. The effects of the variants as well as the resulting cellular iron dyshomeostasis potentially impact a number of disease-associated pathways. We tested the hypothesis that the two most common HFE variants, H63D and C282Y, would affect cellular secretion of cytokines and trophic factors. Methods We screened a panel of cytokines and trophic factors using a multiplexed immunoassay in human neuroblastoma SH-SY5Y cells expressing different variants of HFE. The influence of cellular iron secretion on the potent chemokine monocyte chemoattractant protein-1 (MCP-1 was assessed using ferric ammonium citrate and the iron chelator, desferroxamine. Additionally, an antioxidant, Trolox, and an anti-inflammatory, minocycline, were tested for their effects on MCP-1 secretion in the presence of HFE variants. Results Expression of the HFE variants altered the labile iron pool in SH-SY5Y cells. Of the panel of cytokines and trophic factors analyzed, only the release of MCP-1 was affected by the HFE variants. We further examined the relationship between iron and MCP-1 and found MCP-1 secretion tightly associated with intracellular iron status. A potential direct effect of HFE is considered because, despite having similar levels of intracellular iron, the association between HFE genotype and MCP-1 expression was different for the H63D and C282Y HFE variants. Moreover, HFE genotype was a factor in the effect of minocycline, a multifaceted antibiotic used in treating a number of neurologic conditions associated with inflammation, on MCP-1

  3. Identification of Genetic Variants Linking Protein C and Lipoprotein Metabolism: The ARIC Study (Atherosclerosis Risk in Communities).

    Science.gov (United States)

    Pankow, James S; Tang, Weihong; Pankratz, Nathan; Guan, Weihua; Weng, Lu-Chen; Cushman, Mary; Boerwinkle, Eric; Folsom, Aaron R

    2017-03-01

    Previous studies have identified common genetic variants in 4 chromosomal regions that together account for 14% to 15% of the variance in circulating levels of protein C. To further characterize the genetic architecture of protein C, we obtained denser coverage at some loci, extended investigation of protein C to low-frequency and rare variants, and searched for new associations in genes known to influence protein C. Genetic associations with protein C antigen level were evaluated in ≤10 778 European and 3190 black participants aged 45 to 64 years. Analyses included >26 million autosomal variants available after imputation to the 1000 Genomes reference panel along with additional low-frequency and rare variants directly genotyped using the Illumina ITMAT-Broad-CARe chip and Illumina HumanExome BeadChip. Genome-wide significant associations (PC level in both whites and blacks, reaching genome-wide significance in a meta-analysis combining results from both groups (P=1.4×10(-9)). To further investigate a possible link between lipid metabolism and protein C level, we conducted Mendelian randomization analyses using 185 lipid-related genetic variants as instrumental variables. The results indicated that triglycerides, and possibly low-density lipoprotein cholesterol, influence protein C levels. Discovery of variants influencing circulating protein C levels in the CELSR2-PSRC1-SORT1 region may indicate a novel genetic link between lipoprotein metabolism and hemostasis. © 2017 American Heart Association, Inc.

  4. Alternative splicing of DENND1A, a PCOS candidate gene, generates variant 2.

    Science.gov (United States)

    Tee, Meng Kian; Speek, Mart; Legeza, Balázs; Modi, Bhavi; Teves, Maria Eugenia; McAllister, Janette M; Strauss, Jerome F; Miller, Walter L

    2016-10-15

    Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by hyperandrogenism and metabolic disorders. The excess androgens may be of both ovarian and adrenal origin. PCOS has a strong genetic component, and genome-wide association studies have identified several candidate genes, notably DENND1A, which encodes connecdenn 1, involved in trafficking of endosomes. DENND1A encodes two principal variants, V1 (1009 amino acids) and V2 (559 amino acids). The androgen-producing ovarian theca cells of PCOS women over-express V2. Knockdown of V2 in these cells reduces androgen production, and overexpression of V2 in normal theca cells confers upon them a PCOS phenotype of increased androgen synthesis. We report that human adrenal NCI-H295A cells express V1 and V2 mRNA and that the V2 isoform is produced by exonization of sequences in intron 20, which generates a unique exon 20A, encoding the C-terminus of V2. As in human theca cells from normal women, forced expression of V2 in NCI-H295A cells resulted in increased abundance of CYP17A1 and CYP11A1 mRNAs. We also found genetic variation in the intronic region 330 bp upstream from exon 20A, which could have the potential to drive the selective expression of V2. There was no clear association with these variants with PCOS when we analyzed genomc DNA from normal women and women with PCOS. Using minigene expression vectors in NCI-H295A cells, this variable region did not consistently favor splicing of the V2 transcript. These findings suggest increased V2 expression in PCOS theca cells is not the result of genomic sequence variation in intron 20.

  5. A Disease-Causing Variant in PCNA Disrupts a Promiscuous Protein Binding Site.

    Science.gov (United States)

    Duffy, Caroline M; Hilbert, Brendan J; Kelch, Brian A

    2016-03-27

    The eukaryotic DNA polymerase sliding clamp, proliferating cell nuclear antigen or PCNA, is a ring-shaped protein complex that surrounds DNA to act as a sliding platform for increasing processivity of cellular replicases and for coordinating various cellular pathways with DNA replication. A single point mutation, Ser228Ile, in the human PCNA gene was recently identified to cause a disease whose symptoms resemble those of DNA damage and repair disorders. The mutation lies near the binding site for most PCNA-interacting proteins. However, the structural consequences of the S228I mutation are unknown. Here, we describe the structure of the disease-causing variant, which reveals a large conformational change that dramatically transforms the binding pocket for PCNA client proteins. We show that the mutation markedly alters the binding energetics for some client proteins, while another, p21(CIP1), is only mildly affected. Structures of the disease variant bound to peptides derived from two PCNA partner proteins reveal that the binding pocket can adjust conformation to accommodate some ligands, indicating that the binding site is dynamic and pliable. Our work has implications for the plasticity of the binding site in PCNA and reveals how a disease mutation selectively alters interactions to a promiscuous binding site that is critical for DNA metabolism.

  6. Misfolding, degradation, and aggregation of variant proteins. The molecular pathogenesis of short chain acyl-CoA dehydrogenase (SCAD) deficiency

    DEFF Research Database (Denmark)

    Pedersen, Christina Bak; Bross, P.; Winter, V.S.;

    2003-01-01

    Short chain acyl-CoA dehydrogenase (SCAD) deficiency is an inborn error of the mitochondrial fatty acid metabolism caused by rare variations as well as common susceptibility variations in the SCAD gene. Earlier studies have shown that a common variant SCAD protein (R147W) was impaired in folding...... and aggregation of variant SCAD proteins. In this study we investigated the processing of a set of disease-causing variant SCAD proteins (R22W, G68C, W153R, R359C, and Q341H) and two common variant proteins (R147W and G185S) that lead to reduced SCAD activity. All SCAD proteins, including the wild type, associate...... with mitochondrial hsp60 chaperonins; however, the variant SCAD proteins remained associated with hsp60 for prolonged periods of time. Biogenesis experiments at two temperatures revealed that some of the variant proteins (R22W, G68C, W153R, and R359C) caused severe misfolding, whereas others (R147W, G185S, and Q341H...

  7. New variants of lepidoptericidal toxin genes encoding Bacillus thuringiensis Vip3Aa proteins.

    Science.gov (United States)

    Sauka, Diego H; Rodriguez, Sonia E; Benintende, Graciela B

    2012-01-01

    Bacillus thuringiensis is an entomopathogenic bacterium characterized by producing parasporal proteinaceous insecticidal crystal inclusions during sporulation. Many strains are capable of also expressing other insecticidal proteins called Vip during the vegetative growing phase. Particularly, Vip3A proteins have activity against certain Lepidoptera species through a unique mechanism of action which emphasized their possible use in resistance management strategies against resistant pests. The aim of the work was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that can distinguish between vip3A genes from B. thuringiensis strains. In addition, 4 novel vip3Aa genes were cloned and sequenced. The method was originally based on amplification of a single PCR amplicon and the use of 2 restriction enzymes with recognition sites that facilitate simultaneous detection. Subsequently, a third restriction enzyme was used to distinguish between vip3A variants. Thirteen vip3Aa genes were identified in strains belonging to 10 different B. thuringiensis serovars. Three intra-subclass variants of vip3Aa genes could be differentiated. The presented method can serve as an invaluable tool for the investigation of known and novel vip3A genes in B. thuringiensis strains. To the best of our knowledge, this is the first report where variants of a same subclass of insecticidal genes could be distinguished following PCR-RFLP.

  8. Low density lipoprotein receptor related protein 1 and 6 gene variants and ischaemic stroke risk.

    Science.gov (United States)

    Harriott, A M; Heckman, M G; Rayaprolu, S; Soto-Ortolaza, A I; Diehl, N N; Kanekiyo, T; Liu, C-C; Bu, G; Malik, R; Cole, J W; Meschia, J F; Ross, O A

    2015-08-01

    Low density lipoprotein receptor related proteins (LRPs) 1 and 6 have been implicated in cerebral ischaemia. In addition, genetic variation in LRP1 and LRP6 has been linked with various factors that are related to risk of ischaemic stroke. The aim of this study was to examine the association of LRP1 and LRP6 gene variants with risk of ischaemic stroke as part of the Ischemic Stroke Genetics Study (ISGS). A Caucasian series (434 stroke patients, 319 controls) and an African American series (161 stroke patients, 116 controls) were included. Fourteen LRP6 variants and three LRP1 variants were genotyped and assessed for association with ischaemic stroke. In the Caucasian series, significant associations with ischaemic stroke were observed for LRP6 rs2075241 [odds ratio (OR) 0.42, P = 0.023], rs2302685 (OR 0.44, P = 0.049), rs7975614 (OR 0.07, P = 0.017), rs10492120 (OR 0.62, P = 0.036) and rs10743980 (OR 0.66, P = 0.037). Risk of ischaemic stroke was significantly lower for carriers of any of these five protective LRP6 variants (24.0% of subjects) compared to non-carriers (OR 0.57, P = 0.003). The protective association for LRP6 rs2075241 was observed at a similar magnitude across ischaemic stroke subtypes, whilst the effects of rs23022685, rs10492120 and rs10743980 were most apparent for cardioembolic and large vessel stroke. In the African American series, LRP1 rs11172113 was associated with an increased risk of stroke (OR 1.89, P = 0.006). The results of our preliminary study provide evidence that LRP6 and LRP1 variants may be associated with risk of ischaemic stroke. Validation in larger studies is warranted. © 2015 EAN.

  9. A Human Variant of Glucose-Regulated Protein 94 That Inefficiently Supports IGF Production

    DEFF Research Database (Denmark)

    Marzec, Michal; Hawkes, Colin P; Eletto, Davide

    2016-01-01

    IGFs are critical for normal intrauterine and childhood growth and sustaining health throughout life. We showed previously that the production of IGF-1 and IGF-2 requires interaction with the chaperone glucose-regulated protein 94 (GRP94) and that the amount of secreted IGFs is proportional...... to the GRP94 activity. Therefore, we tested the hypothesis that functional polymorphisms of human GRP94 affect IGF production and thereby human health. We describe a hypomorphic variant of human GRP94, P300L, whose heterozygous carriers have 9% lower circulating IGF-1 concentration. P300L was found first....... Furthermore, recombinant P300L showed impaired nucleotide binding activity. These in vitro data strongly support a causal relationship between the GRP94 variant and the decreased concentration of circulating IGF-1, as observed in human carriers of P300L. Thus, mutations in GRP94 that affect its IGF chaperone...

  10. Identification of cancer predisposition variants in apparently healthy individuals using a next-generation sequencing-based family genomics approach.

    Science.gov (United States)

    Karageorgos, Ioannis; Mizzi, Clint; Giannopoulou, Efstathia; Pavlidis, Cristiana; Peters, Brock A; Zagoriti, Zoi; Stenson, Peter D; Mitropoulos, Konstantinos; Borg, Joseph; Kalofonos, Haralabos P; Drmanac, Radoje; Stubbs, Andrew; van der Spek, Peter; Cooper, David N; Katsila, Theodora; Patrinos, George P

    2015-06-20

    Cancer, like many common disorders, has a complex etiology, often with a strong genetic component and with multiple environmental factors contributing to susceptibility. A considerable number of genomic variants have been previously reported to be causative of, or associated with, an increased risk for various types of cancer. Here, we adopted a next-generation sequencing approach in 11 members of two families of Greek descent to identify all genomic variants with the potential to predispose family members to cancer. Cross-comparison with data from the Human Gene Mutation Database identified a total of 571 variants, from which 47 % were disease-associated polymorphisms, 26 % disease-associated polymorphisms with additional supporting functional evidence, 19 % functional polymorphisms with in vitro/laboratory or in vivo supporting evidence but no known disease association, 4 % putative disease-causing mutations but with some residual doubt as to their pathological significance, and 3 % disease-causing mutations. Subsequent analysis, focused on the latter variant class most likely to be involved in cancer predisposition, revealed two variants of prime interest, namely MSH2 c.2732T>A (p.L911R) and BRCA1 c.2955delC, the first of which is novel. KMT2D c.13895delC and c.1940C>A variants are additionally reported as incidental findings. The next-generation sequencing-based family genomics approach described herein has the potential to be applied to other types of complex genetic disorder in order to identify variants of potential pathological significance.

  11. Impaired protein stability and nuclear localization of NOBOX variants associated with premature ovarian insufficiency.

    Science.gov (United States)

    Ferrari, Ilaria; Bouilly, Justine; Beau, Isabelle; Guizzardi, Fabiana; Ferlin, Alberto; Pollazzon, Marzia; Salerno, Mariacarolina; Binart, Nadine; Persani, Luca; Rossetti, Raffaella

    2016-10-23

    Premature ovarian insufficiency (POI) is a clinical syndrome defined by a loss of ovarian activity before the age of 40. Its pathogenesis is still largely unknown, but increasing evidences support a genetic basis in most cases. Among these, heterozygous mutations in NOBOX, a homeobox gene encoding a transcription factor expressed specifically by oocyte and granulosa cells within the ovary, have been reported in ∼6% of women with sporadic POI. The pivotal role of NOBOX in early folliculogenesis is supported by findings in knock-out mice. Here, we report the genetic screening of 107 European women with idiopathic POI, recruited in various settings, and the molecular and functional characterization of the identified variants to evaluate their involvement in POI onset. Specifically, we report the identification of two novel and two recurrent heterozygous NOBOX variants in 7 out of 107 patients, with a prevalence of 6.5% (upper 95% confidence limit of 11.17%). Furthermore, immunolocalization, Western Blot and transcriptional assays conducted in either HEK293T or CHO cells revealed that all the studied variants (p.R44L, p.G91W, p.G111R, p.G152R, p.K273*, p.R449* and p.D452N) display variable degrees of functional impairment, including defects in transcriptional activity, autophagosomal degradation, nuclear localization or protein instability. Several variants conserve the ability to interact with FOXL2 in intracellular aggregates. Their inability to sustain gene expression, together with their likely aberrant effects on protein stability and degradation, make the identified NOBOX mutations a plausible cause of POI onset.

  12. A Splice Variant of Bardet-Biedl Syndrome 5 (BBS5 Protein that Is Selectively Expressed in Retina.

    Directory of Open Access Journals (Sweden)

    Susan N Bolch

    Full Text Available Bardet-Biedl syndrome is a complex ciliopathy that usually manifests with some form of retinal degeneration, amongst other ciliary-related deficiencies. One of the genetic causes of this syndrome results from a defect in Bardet-Biedl Syndrome 5 (BBS5 protein. BBS5 is one component of the BBSome, a complex of proteins that regulates the protein composition in cilia. In this study, we identify a smaller molecular mass form of BBS5 as a variant formed by alternative splicing and show that expression of this splice variant is restricted to the retina.Reverse transcription PCR from RNA was used to isolate and identify potential alternative transcripts of Bbs5. A peptide unique to the C-terminus of the BBS5 splice variant was synthesized and used to prepare antibodies that selectively recognized the BBS5 splice variant. These antibodies were used on immunoblots of tissue extracts to determine the extent of expression of the alternative transcript and on tissue slices to determine the localization of expressed protein. Pull-down of fluorescently labeled arrestin1 by immunoprecipitation of the BBS5 splice variant was performed to assess functional interaction between the two proteins.PCR from mouse retinal cDNA using Bbs5-specific primers amplified a unique cDNA that was shown to be a splice variant of BBS5 resulting from the use of cryptic splicing sites in Intron 7. The resulting transcript codes for a truncated form of the BBS5 protein with a unique 24 amino acid C-terminus, and predicted 26.5 kD molecular mass. PCR screening of RNA isolated from various ciliated tissues and immunoblots of protein extracts from these same tissues showed that this splice variant was expressed in retina, but not brain, heart, kidney, or testes. Quantitative PCR showed that the splice variant transcript is 8.9-fold (+/- 1.1-fold less abundant than the full-length transcript. In the retina, the splice variant of BBS5 appears to be most abundant in the connecting cilium

  13. Single Nucleotide Variants in the Protein C Pathway and Mortality in Dialysis Patients

    Science.gov (United States)

    Ocak, Gürbey; Drechsler, Christiane; Vossen, Carla Y.; Vos, Hans L.; Rosendaal, Frits R.; Reitsma, Pieter H.; Hoffmann, Michael M.; März, Winfried; Ouwehand, Willem H.; Krediet, Raymond T.; Boeschoten, Elisabeth W.; Dekker, Friedo W.; Wanner, Christoph; Verduijn, Marion

    2014-01-01

    Background The protein C pathway plays an important role in the maintenance of endothelial barrier function and in the inflammatory and coagulant processes that are characteristic of patients on dialysis. We investigated whether common single nucleotide variants (SNV) in genes encoding protein C pathway components were associated with all-cause 5 years mortality risk in dialysis patients. Methods Single nucleotides variants in the factor V gene (F5 rs6025; factor V Leiden), the thrombomodulin gene (THBD rs1042580), the protein C gene (PROC rs1799808 and 1799809) and the endothelial protein C receptor gene (PROCR rs867186, rs2069951, and rs2069952) were genotyped in 1070 dialysis patients from the NEtherlands COoperative Study on the Adequacy of Dialysis (NECOSAD) cohort) and in 1243 dialysis patients from the German 4D cohort. Results Factor V Leiden was associated with a 1.5-fold (95% CI 1.1–1.9) increased 5-year all-cause mortality risk and carriers of the AG/GG genotypes of the PROC rs1799809 had a 1.2-fold (95% CI 1.0–1.4) increased 5-year all-cause mortality risk. The other SNVs in THBD, PROC, and PROCR were not associated with 5-years mortality. Conclusion Our study suggests that factor V Leiden and PROC rs1799809 contributes to an increased mortality risk in dialysis patients. PMID:24816905

  14. Coval: Improving Alignment Quality and Variant Calling Accuracy for Next-Generation Sequencing Data

    Science.gov (United States)

    Kosugi, Shunichi; Natsume, Satoshi; Yoshida, Kentaro; MacLean, Daniel; Cano, Liliana; Kamoun, Sophien; Terauchi, Ryohei

    2013-01-01

    Accurate identification of DNA polymorphisms using next-generation sequencing technology is challenging because of a high rate of sequencing error and incorrect mapping of reads to reference genomes. Currently available short read aligners and DNA variant callers suffer from these problems. We developed the Coval software to improve the quality of short read alignments. Coval is designed to minimize the incidence of spurious alignment of short reads, by filtering mismatched reads that remained in alignments after local realignment and error correction of mismatched reads. The error correction is executed based on the base quality and allele frequency at the non-reference positions for an individual or pooled sample. We demonstrated the utility of Coval by applying it to simulated genomes and experimentally obtained short-read data of rice, nematode, and mouse. Moreover, we found an unexpectedly large number of incorrectly mapped reads in ‘targeted’ alignments, where the whole genome sequencing reads had been aligned to a local genomic segment, and showed that Coval effectively eliminated such spurious alignments. We conclude that Coval significantly improves the quality of short-read sequence alignments, thereby increasing the calling accuracy of currently available tools for SNP and indel identification. Coval is available at http://sourceforge.net/projects/coval105/. PMID:24116042

  15. Coval: improving alignment quality and variant calling accuracy for next-generation sequencing data.

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    Shunichi Kosugi

    Full Text Available Accurate identification of DNA polymorphisms using next-generation sequencing technology is challenging because of a high rate of sequencing error and incorrect mapping of reads to reference genomes. Currently available short read aligners and DNA variant callers suffer from these problems. We developed the Coval software to improve the quality of short read alignments. Coval is designed to minimize the incidence of spurious alignment of short reads, by filtering mismatched reads that remained in alignments after local realignment and error correction of mismatched reads. The error correction is executed based on the base quality and allele frequency at the non-reference positions for an individual or pooled sample. We demonstrated the utility of Coval by applying it to simulated genomes and experimentally obtained short-read data of rice, nematode, and mouse. Moreover, we found an unexpectedly large number of incorrectly mapped reads in 'targeted' alignments, where the whole genome sequencing reads had been aligned to a local genomic segment, and showed that Coval effectively eliminated such spurious alignments. We conclude that Coval significantly improves the quality of short-read sequence alignments, thereby increasing the calling accuracy of currently available tools for SNP and indel identification. Coval is available at http://sourceforge.net/projects/coval105/.

  16. Estimating the effect of human base excision repair protein variants on the repair of oxidative DNA base damage.

    Science.gov (United States)

    Sokhansanj, Bahrad A; Wilson, David M

    2006-05-01

    Epidemiologic studies have revealed a complex association between human genetic variance and cancer risk. Quantitative biological modeling based on experimental data can play a critical role in interpreting the effect of genetic variation on biochemical pathways relevant to cancer development and progression. Defects in human DNA base excision repair (BER) proteins can reduce cellular tolerance to oxidative DNA base damage caused by endogenous and exogenous sources, such as exposure to toxins and ionizing radiation. If not repaired, DNA base damage leads to cell dysfunction and mutagenesis, consequently leading to cancer, disease, and aging. Population screens have identified numerous single-nucleotide polymorphism variants in many BER proteins and some have been purified and found to exhibit mild kinetic defects. Epidemiologic studies have led to conflicting conclusions on the association between single-nucleotide polymorphism variants in BER proteins and cancer risk. Using experimental data for cellular concentration and the kinetics of normal and variant BER proteins, we apply a previously developed and tested human BER pathway model to (i) estimate the effect of mild variants on BER of abasic sites and 8-oxoguanine, a prominent oxidative DNA base modification, (ii) identify ranges of variation associated with substantial BER capacity loss, and (iii) reveal nonintuitive consequences of multiple simultaneous variants. Our findings support previous work suggesting that mild BER variants have a minimal effect on pathway capacity whereas more severe defects and simultaneous variation in several BER proteins can lead to inefficient repair and potentially deleterious consequences of cellular damage.

  17. Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics.

    Science.gov (United States)

    Aliye, Naser; Fabbretti, Attilio; Lupidi, Giulio; Tsekoa, Tsepo; Spurio, Roberto

    2015-02-01

    A number of studies have been conducted to improve chromophore maturation, folding kinetics, thermostability, and other traits of green fluorescent protein (GFP). However, no specific work aimed at improving the thermostability of the yellow fluorescent protein (YFP) and of the pH-sensitive, yet thermostable color variants of GFP has so far been done. The protein variants reported in this study were improved through rational multiple site-directed mutagenesis of GFP (ASV) by introducing up to ten point mutations including the mutations near and at the chromophore region. Therefore, we report the development and characterization of fast folder and thermo-tolerant green variant (FF-GFP), and a fast folder thermostable yellow fluorescent protein (FFTS-YFP) endowed with remarkably improved thermostability and folding kinetics. We demonstrate that the fluorescence intensity of this yellow variant is not affected by heating at 75 °C. Moreover, we have developed a pH-unresponsive cyan variant AcS-CFP, which has potential use as part of in vivo imaging irrespective of intracellular pH. The combined improved properties make these fluorescent variants ideal tools to study protein expression and function under different pH environments, in mesophiles and thermophiles. Furthermore, coupling of the FFTS-YFP and AcS-CFP could potentially serve as an ideal tool to perform functional analysis of live cells by multicolor labeling.

  18. Molecular analysis and physicochemical properties of electrophoretic variants of wild soybean Glycine soja storage proteins.

    Science.gov (United States)

    Fukuda, Takako; Maruyama, Nobuyuki; Kanazawa, Akira; Abe, Jun; Shimamoto, Yoshiya; Hiemori, Miki; Tsuji, Hideaki; Tanisaka, Takatoshi; Utsumi, Shigeru

    2005-05-04

    Cultivated soybeans (Glycine max) are derived from wild soybeans (Glycine soja) and can be crossed with them to produce fertile offspring. The latter exhibit greater genetic variation than the former, suggesting a possibility that wild soybeans contain storage proteins with properties different from and better than those of cultivated soybeans. To identify a wild soybean suitable for breeding a new soybean cultivar, we analyzed seed proteins from 390 lines of wild soybeans by electrophoresis. We found some lines containing electrophoretic variants of glycinin and beta-conglycinin subunits: one line containing a small alpha' subunit of beta-conglycinin and two and five lines containing small A3 and large A4 polypeptides of glycinin, respectively. Beta-Conglycinin and glycinin containing such variant subunits exhibited solubility and emulsifying ability similar to those of the predominant types of wild and cultivated soybeans. Glycinins containing small A3 and large A4 gave a shoulder derived from the start of denaturation at a temperature 4 degrees C lower than that of glycinin from the predominant types of wild and cultivated soybeans, although their thermal denaturation midpoint temperatures were very similar to each other. Cloning and sequencing of the predominant and variant subunit cDNAs revealed that the small alpha' and the small A3 lacked 24 amino acid residues in the extension region and four amino acid residues in the hypervariable region, respectively, and that the large A4 did not have an insert corresponding to the difference in the electrophoretic mobility but Arg279 and Gln305 were replaced by glutamine and histidine, respectively, in the hypervariable region. These suggest that small differences even in the hypervariable region can affect the thermal stability, as well as the electrophoretic mobilities, of the proteins.

  19. Myosin-binding Protein C Compound Heterozygous Variant Effect on the Phenotypic Expression of Hypertrophic Cardiomyopathy

    Science.gov (United States)

    Rafael, Julianny Freitas; Cruz Filho, Fernando Eugênio dos Santos; de Carvalho, Antônio Carlos Campos; Gottlieb, Ilan; Cazelli, José Guilherme; Siciliano, Ana Paula; Dias, Glauber Monteiro

    2017-01-01

    Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disease caused by mutations in genes encoding sarcomere proteins. It is the major cause of sudden cardiac death in young high-level athletes. Studies have demonstrated a poorer prognosis when associated with specific mutations. The association between HCM genotype and phenotype has been the subject of several studies since the discovery of the genetic nature of the disease. This study shows the effect of a MYBPC3 compound variant on the phenotypic HCM expression. A family in which a young man had a clinical diagnosis of HCM underwent clinical and genetic investigations. The coding regions of the MYH7, MYBPC3 and TNNT2 genes were sequenced and analyzed. The proband present a malignant manifestation of the disease, and is the only one to express HCM in his family. The genetic analysis through direct sequencing of the three main genes related to this disease identified a compound heterozygous variant (p.E542Q and p.D610H) in MYBPC3. A family analysis indicated that the p.E542Q and p.D610H alleles have paternal and maternal origin, respectively. No family member carrier of one of the variant alleles manifested clinical signs of HCM. We suggest that the MYBPC3-biallelic heterozygous expression of p.E542Q and p.D610H may cause the severe disease phenotype seen in the proband. PMID:28538763

  20. Genome-Wide Functional Annotation of Human Protein-Coding Splice Variants Using Multiple Instance Learning.

    Science.gov (United States)

    Panwar, Bharat; Menon, Rajasree; Eksi, Ridvan; Li, Hong-Dong; Omenn, Gilbert S; Guan, Yuanfang

    2016-06-03

    The vast majority of human multiexon genes undergo alternative splicing and produce a variety of splice variant transcripts and proteins, which can perform different functions. These protein-coding splice variants (PCSVs) greatly increase the functional diversity of proteins. Most functional annotation algorithms have been developed at the gene level; the lack of isoform-level gold standards is an important intellectual limitation for currently available machine learning algorithms. The accumulation of a large amount of RNA-seq data in the public domain greatly increases our ability to examine the functional annotation of genes at isoform level. In the present study, we used a multiple instance learning (MIL)-based approach for predicting the function of PCSVs. We used transcript-level expression values and gene-level functional associations from the Gene Ontology database. A support vector machine (SVM)-based 5-fold cross-validation technique was applied. Comparatively, genes with multiple PCSVs performed better than single PCSV genes, and performance also improved when more examples were available to train the models. We demonstrated our predictions using literature evidence of ADAM15, LMNA/C, and DMXL2 genes. All predictions have been implemented in a web resource called "IsoFunc", which is freely available for the global scientific community through http://guanlab.ccmb.med.umich.edu/isofunc .

  1. Impaired suppressive activities of human MUTYH variant proteins against oxidative mutagenesis

    Institute of Scientific and Technical Information of China (English)

    Kazuya Shinmura; Masanori Goto; Hong Tao; Shun Matsuura; Tomonari Matsuda; Haruhiko Sugimura

    2012-01-01

    AIM:To investigate the suppressive activity of MUTYH variant proteins against mutations caused by oxidative lesion,8-hydroxyguanine (8OHG),in human cells.METHODS:p.R154H,p.M255V,p.L360P,and p.P377L MUTYH variants,which were previously found in patients with colorectal polyposis and cancer,were selected for use in this study.Human H1299 cancer cell lines inducibly expressing wild-type (WT) MUTYH (type 2) or one of the 4 above-mentioned MUTYH variants were established using the piggyBac transposon vector system,enabling the genomic integration of the transposon sequence for MUTYH expression.MUTYH expression was examined after cumate induction using Western blotting analysis and immunofiuorescence analysis.The intracellular localization of MUTYH variants tagged with FLAG was also immunofluorescently examined.Next,the mutation frequency in the supF of the shuttle plasmid pMY189 containing a single 8OHG residue at position 159 of the supFwas compared between empty vector cells and cells expressing WT MUTYH or one of the 4 MUTYH variants using a supF forward mutation assay.RESULTS:The successful establishment of human cell lines inducibly expressing WT MUTYH or one of the 4 MUTYH variants was concluded based on the detection of MUTYH expression in these cell lines after treatment with cumate.All of the MUTYH variants and WT MUTYH were localized in the nucleus,and nuclear localization was also observed for FLAG-tagged MUTYH.The mutation frequency ofsupFwas 2.2 x 102in the 8OHG-containing pMY189 plasmid and 2.5× 10-4 in WT pMY189 in empty vector cells,which was an 86-fold increase with the introduction of 8OHG.The mutation frequency (4.7 × 10-3) of supF in the 8OHG-containing pMY189 plasmid in cells overexpressing WT MUTYH was significantly lower than in the empty vector cells (P < 0.01).However,the mutation frequencies of the supF in the 8OHG-containing pMY189 plasmid in cells overexpressing the p.R154H,p.M255V,p.L360P,or p.P377L MUTYH variant were 1.84 × 10-2,1.55

  2. Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

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    O Rickard Nilsson

    Full Text Available Recent studies indicate that defective activity of complement factor H (FH is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

  3. Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

    Science.gov (United States)

    Nilsson, O Rickard; Lannergård, Jonas; Morgan, B Paul; Lindahl, Gunnar; Gustafsson, Mattias C U

    2013-01-01

    Recent studies indicate that defective activity of complement factor H (FH) is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR) of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD) and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

  4. Re-fraction: a machine learning approach for deterministic identification of protein homologues and splice variants in large-scale MS-based proteomics.

    Science.gov (United States)

    Yang, Pengyi; Humphrey, Sean J; Fazakerley, Daniel J; Prior, Matthew J; Yang, Guang; James, David E; Yang, Jean Yee-Hwa

    2012-05-04

    A key step in the analysis of mass spectrometry (MS)-based proteomics data is the inference of proteins from identified peptide sequences. Here we describe Re-Fraction, a novel machine learning algorithm that enhances deterministic protein identification. Re-Fraction utilizes several protein physical properties to assign proteins to expected protein fractions that comprise large-scale MS-based proteomics data. This information is then used to appropriately assign peptides to specific proteins. This approach is sensitive, highly specific, and computationally efficient. We provide algorithms and source code for the current version of Re-Fraction, which accepts output tables from the MaxQuant environment. Nevertheless, the principles behind Re-Fraction can be applied to other protein identification pipelines where data are generated from samples fractionated at the protein level. We demonstrate the utility of this approach through reanalysis of data from a previously published study and generate lists of proteins deterministically identified by Re-Fraction that were previously only identified as members of a protein group. We find that this approach is particularly useful in resolving protein groups composed of splice variants and homologues, which are frequently expressed in a cell- or tissue-specific manner and may have important biological consequences.

  5. Vitamin D binding protein variants associate with asthma susceptibility in the Chinese han population

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    Zhang Youming

    2011-08-01

    Full Text Available Abstract Background Asthma is a genetically heterogeneous disease. Polymorphisms of genes encoding components of the vitamin D pathway have been reported to associate with the risk of asthma. We have previously demonstrated that vitamin D status was associated with lung function in Chinese asthma patients. In this study, we tested whether polymorphisms of genes encoding for vitamin D receptor (VDR, vitamin D 25-hydroxylase (CYP2R1 and vitamin D binding protein (GC were associated with asthma in the Chinese Han population. Methods We sequenced all 8 exons of VDR and all 5 exons of CYP2R1 in a Chinese case-control cohort of asthma consisting of 467 cases and 288 unrelated healthy controls. Two mutations were identified in these regions. These variants were specified as rs2228570 in exon 2 of VDR and rs12794714 in exon 1 of CYP2R1. We also genotyped two common polymorphisms in GC gene (rs4588 and rs7041 by a PCR-restriction fragment length polymorphism (RFLP method. We analyzed the association between these 4 polymorphisms and asthma susceptibility and asthma-related traits. Results Polymorphic markers in VDR and CYP2R1 were not associated with asthma in the Chinese Han cohort. Importantly, variants in GC gene, which give rise to the two most common electrophoretic isoforms of the vitamin D binding protein, were associated with asthma susceptibility. Compared with isoform Gc1, Gc2 was significantly associated with the risk of asthma (OR = 1.35, 95% CI = 1.01-1.78 p = 0.006. Conclusions The results provide supporting evidence for association between GC variants and asthma susceptibility in the Chinese Han population.

  6. Quantitative mass spectrometry evaluation of human retinol binding protein 4 and related variants.

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    Urban A Kiernan

    Full Text Available BACKGROUND: Retinol Binding Protein 4 (RBP4 is an exciting new biomarker for the determination of insulin resistance and type 2 diabetes. It is known that circulating RBP4 resides in multiple variants which may provide enhanced clinical utility, but conventional immunoassay methods are blind to such differences. A Mass Spectrometric immunoassay (MSIA technology that can quantitate total RBP4 as well as individual isoforms may provide an enhanced analysis for this biomarker. METHODS: RBP4 was isolated and detected from 0.5 uL of human plasma using MSIA technology, for the simultaneous quantification and differentiation of endogenous human RBP4 and its variants. RESULTS: The linear range of the assay was 7.81-500 ug/mL, and the limit of detection and limit of quantification were 3.36 ug/mL and 6.52 ug/mL, respectively. The intra-assay CVs were determined to be 5.1% and the inter-assay CVs were 9.6%. The percent recovery of the RBP4-MSIA ranged from 95 - 105%. Method comparison of the RBP4 MSIA vs the Immun Diagnostik ELISA yielded a Passing & Bablok fit of MSIA  = 1.05× ELISA - 3.09, while the Cusum linearity p-value was >0.1 and the mean bias determined by the Altman Bland test was 1.2%. CONCLUSION: The novel RBP4 MSIA provided a fast, accurate and precise quantitative protein measurement as compared to the standard commercially available ELISA. Moreover, this method also allowed for the detection of RBP4 variants that are present in each sample, which may in the future provide a new dimension in the clinical utility of this biomarker.

  7. Separation of monoclonal antibody charge state variants by open tubular capillary electrochromatography with immobilised protein as stationary phase.

    Science.gov (United States)

    Zhang, Yamin; Wang, Wentao; Xiao, Xue; Jia, Li

    2016-09-30

    Monoclonal antibodies (mAbs) are highly heterogeneous and complex glycoproteins requiring powerful analytical tools for characterization and quality control. In this work, we utilize adsorbed bovine serum albumin (BSA) as a stationary phase in open tubular (OT) capillary electrochromatography for separation of charge state variants of mAbs. Poly(diallydimethylammonium chloride) (PDDA) was used to assist fabrication of BSA coated OT column by electrostatic self-assembly. Scanning electron microscopy and electroosmotic flow measurement were carried out to characterize the as-prepared BSA coated PDDA OT columns. The electrochromatographic performance of the OT columns was evaluated by separation of basic proteins and different charge state variants of mAbs. The effects of background solution pH and concentration on separation were investigated. A rapid separation of charge state variants of mAbs was successfully achieved in the BSA coated PDDA OT column. Separation of seven variants of the mAb cetuximab was achieved using the prepared column. Two basic variants and one acidic variant of rituximab, and two basic variants and four acidic variants of trastuximab were successfully distinguished from the main forms. In addition, the columns demonstrated good repeatability and stability with the run-to-run, day-to-day and batch-to-batch relative standard deviations of migration times less than 3.7%.

  8. Structure-function analysis of two variants of mumps virus hemagglutinin-neuraminidase protein

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    Gerardo Santos-López

    2009-02-01

    Full Text Available A point mutation from guanine (G to adenine (A at nucleotide position 1081 in the hemagglutinin-neuraminidase (HN gene has been associated with neurovirulence of Urabe AM9 mumps virus vaccine. This mutation corresponds to a glutamic acid (E to lysine (K change at position 335 in the HN glycoprotein. We have experimentally demonstrated that two variants of Urabe AM9 strain (HN-A1081 and HN-G1081 differ in neurotropism, sialic acidbinding affinity and neuraminidase activity. In the present study, we performed a structure-function analysis of that amino acid substitution; the structures of HN protein of both Urabe AM9 strain variants were predicted. Based on our analysis, the E/K mutation changes the protein surface properties and to a lesser extent their conformations, which in turn reflects in activity changes. Our modeling results suggest that this E/K interchange does not affect the structure of the sialic acid binding motif; however, the electrostatic surface differs drastically due to an exposed short alpha helix. Consequently, this mutation may affect the accessibility of HN to substrates and membrane receptors of the host cells. Our findings appear to explain the observed differences in neurotropism of these vaccine strains.

  9. Protein Profiling and Histone Deacetylation Activities in Somaclonal Variants of Oil Palm (Elaeis guineensis Jacq.

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    Jamilah Syafawati Yaacob

    2013-01-01

    Full Text Available Mantled fruits as a result of somaclonal variation are often observed from the oil palm plantlets regenerated via tissue culture. The mantling of fruits with finger-like and thick outer coating phenotypes significantly reduces the seed size and oil content, posing a threat to oil palm planters, and may jeopardize the economic growth of countries that depend particularly on oil palm plantation. The molecular aspects of the occurrence of somaclonal variations are yet to be known, possibly due to gene repression such as DNA methylation, histone methylation and histone deacetylation. Histone deacetylases (HDACs, involved in eukaryotic gene regulation by catalyzing the acetyl groups are removal from lysine residues on histone, hence transcriptionally repress gene expression. This paper described the total protein polymorphism profiles of somaclonal variants of oil palm and the effects of histone deacetylation on this phenomenon. Parallel to the different phenotypes, the protein polymorphism profiles of the mantled samples (leaves, fruits, and florets and the phenotypically normal samples were proven to be different. Higher HDAC activity was found in mantled leaf samples than in the phenotypically normal leaf samples, leading to a preliminary conclusion that histone deacetylation suppressed gene expression and contributed to the development of somaclonal variants.

  10. Protein profiling and histone deacetylation activities in somaclonal variants of oil palm (Elaeis guineensis Jacq.).

    Science.gov (United States)

    Yaacob, Jamilah Syafawati; Loh, Hwei-San; Mat Taha, Rosna

    2013-01-01

    Mantled fruits as a result of somaclonal variation are often observed from the oil palm plantlets regenerated via tissue culture. The mantling of fruits with finger-like and thick outer coating phenotypes significantly reduces the seed size and oil content, posing a threat to oil palm planters, and may jeopardize the economic growth of countries that depend particularly on oil palm plantation. The molecular aspects of the occurrence of somaclonal variations are yet to be known, possibly due to gene repression such as DNA methylation, histone methylation and histone deacetylation. Histone deacetylases (HDACs), involved in eukaryotic gene regulation by catalyzing the acetyl groups are removal from lysine residues on histone, hence transcriptionally repress gene expression. This paper described the total protein polymorphism profiles of somaclonal variants of oil palm and the effects of histone deacetylation on this phenomenon. Parallel to the different phenotypes, the protein polymorphism profiles of the mantled samples (leaves, fruits, and florets) and the phenotypically normal samples were proven to be different. Higher HDAC activity was found in mantled leaf samples than in the phenotypically normal leaf samples, leading to a preliminary conclusion that histone deacetylation suppressed gene expression and contributed to the development of somaclonal variants.

  11. Structural and Functional Characterization of the VQ Protein Family and VQ Protein Variants from Soybean

    Science.gov (United States)

    Zhou, Yuan; Yang, Yan; Zhou, Xinjian; Chi, Yingjun; Fan, Baofang; Chen, Zhixiang

    2016-01-01

    Proteins containing the FxxxVQxhTG or VQ motif interact with WRKY transcription factors. Although VQ proteins have been reported in several plants, knowledge about their structures, functions and evolution is still very limited. Here, we report structural and functional analysis of the VQ protein family from soybean. Like Arabidopsis homologues, soybean VQ proteins bind only Group I and IIc WRKY proteins and a substantial number of their genes are responsive to stress-associated phytohormones. Overexpression of some soybean VQ genes in Arabidopsis had strong effects on plant growth, development, disease resistance and heat tolerance. Phylogenetic analysis, sequence alignment and site-directed mutagenesis revealed that the region immediately upstream of the FxxxVQxhTG motif also affects binding to WRKY proteins. Consistent with a larger WRKY-binding VQ domain, soybean VQ22 protein from cultivated soybean contains a 4-amino acid deletion in the region preceding its VQ motif that completely abolishes its binding to WRKY proteins. By contrast, the 4-amino acid deletion is absent in the VQ22 protein from wild soybean species (Glycine soja). Overexpression of wild soybean VQ22 in cultivated soybean inhibited growth, particularly after cold treatment. Thus, the mutation of soybean VQ22 is associated with advantageous phenotypes and may have been positively selected during evolution. PMID:27708406

  12. Intact Protein Analysis at 21 Tesla and X-Ray Crystallography Define Structural Differences in Single Amino Acid Variants of Human Mitochondrial Branched-Chain Amino Acid Aminotransferase 2 (BCAT2)

    Science.gov (United States)

    Anderson, Lissa C.; Håkansson, Maria; Walse, Björn; Nilsson, Carol L.

    2017-09-01

    Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural coordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a 45 kDa protein, we applied combined ETD and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X-Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modeling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein (PDB code: 5MPR), leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM. [Figure not available: see fulltext.

  13. Intact Protein Analysis at 21 Tesla and X-Ray Crystallography Define Structural Differences in Single Amino Acid Variants of Human Mitochondrial Branched-Chain Amino Acid Aminotransferase 2 (BCAT2)

    Science.gov (United States)

    Anderson, Lissa C.; Håkansson, Maria; Walse, Björn; Nilsson, Carol L.

    2017-07-01

    Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural coordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a 45 kDa protein, we applied combined ETD and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X-Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modeling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein (PDB code: 5MPR), leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM.

  14. Generation of Protein Nanogradients by Microcontact Printing

    Science.gov (United States)

    Schwaab, Daniel; Zentis, Peter; Winter, Silke; Meffert, Simone; Offenhäusser, Andreas; Mayer, Dirk

    2013-05-01

    High resolution lithography combined with microcontact printing (µCP) by means of polyolefine polymer (POP) stamps enabled to create protein gradient patterns. By this means, discrete purely biochemical gradients of extracellular matrix proteins were fabricated. It was possible to adjust independently both the size of elements of a protein pattern and the distance between them with sub 100 nm resolution. Adhesion of primary neurons and directed neuronal outgrowth were observed on these protein patterns. Cellular constituents such as filopodia adhere to different printed protein elements of the discontinuous gradient including features as small as 75 nm.

  15. Viral population analysis and minority-variant detection using short read next-generation sequencing.

    Science.gov (United States)

    Watson, Simon J; Welkers, Matthijs R A; Depledge, Daniel P; Coulter, Eve; Breuer, Judith M; de Jong, Menno D; Kellam, Paul

    2013-03-19

    RNA viruses within infected individuals exist as a population of evolutionary-related variants. Owing to evolutionary change affecting the constitution of this population, the frequency and/or occurrence of individual viral variants can show marked or subtle fluctuations. Since the development of massively parallel sequencing platforms, such viral populations can now be investigated to unprecedented resolution. A critical problem with such analyses is the presence of sequencing-related errors that obscure the identification of true biological variants present at low frequency. Here, we report the development and assessment of the Quality Assessment of Short Read (QUASR) Pipeline (http://sourceforge.net/projects/quasr) specific for virus genome short read analysis that minimizes sequencing errors from multiple deep-sequencing platforms, and enables post-mapping analysis of the minority variants within the viral population. QUASR significantly reduces the error-related noise in deep-sequencing datasets, resulting in increased mapping accuracy and reduction of erroneous mutations. Using QUASR, we have determined influenza virus genome dynamics in sequential samples from an in vitro evolution of 2009 pandemic H1N1 (A/H1N1/09) influenza from samples sequenced on both the Roche 454 GSFLX and Illumina GAIIx platforms. Importantly, concordance between the 454 and Illumina sequencing allowed unambiguous minority-variant detection and accurate determination of virus population turnover in vitro.

  16. Novel microcephalic primordial dwarfism disorder associated with variants in the centrosomal protein ninein.

    Science.gov (United States)

    Dauber, Andrew; Lafranchi, Stephen H; Maliga, Zoltan; Lui, Julian C; Moon, Jennifer E; McDeed, Cailin; Henke, Katrin; Zonana, Jonathan; Kingman, Garrett A; Pers, Tune H; Baron, Jeffrey; Rosenfeld, Ron G; Hirschhorn, Joel N; Harris, Matthew P; Hwa, Vivian

    2012-11-01

    Microcephalic primordial dwarfism (MPD) is a rare, severe form of human growth failure in which growth restriction is evident in utero and continues into postnatal life. Single causative gene defects have been identified in a number of patients with MPD, and all involve genes fundamental to cellular processes including centrosome functions. The objective of the study was to find the genetic etiology of a novel presentation of MPD. The design of the study was whole-exome sequencing performed on two affected sisters in a single family. Molecular and functional studies of a candidate gene were performed using patient-derived primary fibroblasts and a zebrafish morpholino oligonucleotides knockdown model. Two sisters presented with a novel subtype of MPD, including severe intellectual disabilities. NIN, encoding Ninein, a centrosomal protein critically involved in asymmetric cell division, was identified as a candidate gene, and functional impacts in fibroblasts and zebrafish were studied. From 34,606 genomic variants, two very rare missense variants in NIN were identified. Both probands were compound heterozygotes. In the zebrafish, ninein knockdown led to specific and novel defects in the specification and morphogenesis of the anterior neuroectoderm, resulting in a deformity of the developing cranium with a small, squared skull highly reminiscent of the human phenotype. We identified a novel clinical subtype of MPD in two sisters who have rare variants in NIN. We show, for the first time, that reduction of ninein function in the developing zebrafish leads to specific deficiencies of brain and skull development, offering a developmental basis for the myriad phenotypes in our patients.

  17. Generation of novel AAV variants by directed evolution for improved CFTR delivery to human ciliated airway epithelium.

    Science.gov (United States)

    Li, Wuping; Zhang, Liqun; Johnson, Jarrod S; Zhijian, Wu; Grieger, Joshua C; Ping-Jie, Xiao; Drouin, Lauren M; Agbandje-McKenna, Mavis; Pickles, Raymond J; Samulski, R Jude

    2009-12-01

    Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.

  18. Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

    Science.gov (United States)

    Li, Wuping; Zhang, Liqun; Johnson, Jarrod S; Zhijian, Wu; Grieger, Joshua C; Ping-Jie, Xiao; Drouin, Lauren M; Agbandje-McKenna, Mavis; Pickles, Raymond J; Samulski, R Jude

    2009-01-01

    Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease. PMID:19603002

  19. Pancreatic cancer cells express CD44 variant 9 and multidrug resistance protein 1 during mitosis.

    Science.gov (United States)

    Kiuchi, Shizuka; Ikeshita, Shunji; Miyatake, Yukiko; Kasahara, Masanori

    2015-02-01

    Pancreatic cancer is one of the most lethal cancers with high metastatic potential and strong chemoresistance. Its intractable natures are attributed to high robustness in tumor cells for their survival. We demonstrate here that pancreatic cancer cells (PCCs) with an epithelial phenotype upregulate cell surface expression of CD44 variant 9 (CD44v9), an important cancer stem cell marker, during the mitotic phases of the cell cycle. Of five human CD44(+) PCC lines examined, three cell lines, PCI-24, PCI-43 and PCI-55, expressed E-cadherin and CD44 variants, suggesting that they have an epithelial phenotype. By contrast, PANC-1 and MIA PaCa-2 cells expressed vimentin and ZEB1, suggesting that they have a mesenchymal phenotype. PCCs with an epithelial phenotype upregulated cell surface expression of CD44v9 in prophase, metaphase, anaphase and telophase and downregulated CD44v9 expression in late-telophase, cytokinesis and interphase. Sorted CD44v9-negative PCI-55 cells resumed CD44v9 expression when they re-entered the mitotic stage. Interestingly, CD44v9(bright) mitotic cells expressed multidrug resistance protein 1 (MDR1) intracellularly. Upregulated expression of CD44v9 and MDR1 might contribute to the intractable nature of PCCs with high proliferative activity.

  20. Conformational diversity in prion protein variants influences intermolecular [beta]-sheet formation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seungjoo; Antony, Lizamma; Hartmann, Rune; Knaus, Karen J.; Surewicz, Krystyna; Surewicz, Witold K.; Yee, Vivien C. (Case Western); (Cleveland Clinic)

    2010-04-19

    A conformational transition of normal cellular prion protein (PrP{sup C}) to its pathogenic form (PrP{sup Sc}) is believed to be a central event in the transmission of the devastating neurological diseases known as spongiform encephalopathies. The common methionine/valine polymorphism at residue 129 in the PrP influences disease susceptibility and phenotype. We report here seven crystal structures of human PrP variants: three of wild-type (WT) PrP containing V129, and four of the familial variants D178N and F198S, containing either M129 or V129. Comparison of these structures with each other and with previously published WT PrP structures containing M129 revealed that only WT PrPs were found to crystallize as domain-swapped dimers or closed monomers; the four mutant PrPs crystallized as non-swapped dimers. Three of the four mutant PrPs aligned to form intermolecular {beta}-sheets. Several regions of structural variability were identified, and analysis of their conformations provides an explanation for the structural features, which can influence the formation and conformation of intermolecular {beta}-sheets involving the M/V129 polymorphic residue.

  1. Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Heon; Kang, Sukmo [College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul (Korea, Republic of); Dong, Mi Sook [School of Life Sciences and Biotechnology, Korea University, Seoul (Korea, Republic of); Park, Jung-Duck [College of Medicine, Chung-Ang University, Seoul (Korea, Republic of); Park, Jinseo; Rhee, Sangkee [College of Agriculture of Life Science, Seoul National University, Seoul (Korea, Republic of); Ryu, Doug-Young, E-mail: dyryu@snu.ac.kr [College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul (Korea, Republic of)

    2015-06-15

    Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E. coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the two regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located. - Highlights: • Ala62 is located in α-helix A of the carcinogen-metabolizing enzyme CYP1A1. • Pro acts as an α-helix breaker. • A variant protein of CYP1A1, Ala62Pro, had lower heme content than the wild-type. • The variant of CYP1A1 had lower enzyme activities than the wild-type.

  2. Differential regulation of protein tyrosine kinase signalling by Dock and the PTP61F variants.

    Science.gov (United States)

    Willoughby, Lee F; Manent, Jan; Allan, Kirsten; Lee, Han; Portela, Marta; Wiede, Florian; Warr, Coral; Meng, Tzu-Ching; Tiganis, Tony; Richardson, Helena E

    2017-07-01

    Tyrosine phosphorylation-dependent signalling is coordinated by the opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). There is a growing list of adaptor proteins that interact with PTPs and facilitate the dephosphorylation of substrates. The extent to which any given adaptor confers selectivity for any given substrate in vivo remains unclear. Here we have taken advantage of Drosophila melanogaster as a model organism to explore the influence of the SH3/SH2 adaptor protein Dock on the abilities of the membrane (PTP61Fm)- and nuclear (PTP61Fn)-targeted variants of PTP61F (the Drosophila othologue of the mammalian enzymes PTP1B and TCPTP respectively) to repress PTK signalling pathways in vivo. PTP61Fn effectively repressed the eye overgrowth associated with activation of the epidermal growth factor receptor (EGFR), PTK, or the expression of the platelet-derived growth factor/vascular endothelial growth factor receptor (PVR) or insulin receptor (InR) PTKs. PTP61Fn repressed EGFR and PVR-induced mitogen-activated protein kinase signalling and attenuated PVR-induced STAT92E signalling. By contrast, PTP61Fm effectively repressed EGFR- and PVR-, but not InR-induced tissue overgrowth. Importantly, coexpression of Dock with PTP61F allowed for the efficient repression of the InR-induced eye overgrowth, but did not enhance the PTP61Fm-mediated inhibition of EGFR and PVR-induced signalling. Instead, Dock expression increased, and PTP61Fm coexpression further exacerbated the PVR-induced eye overgrowth. These results demonstrate that Dock selectively enhances the PTP61Fm-mediated attenuation of InR signalling and underscores the specificity of PTPs and the importance of adaptor proteins in regulating PTP function in vivo. © 2017 Federation of European Biochemical Societies.

  3. Primary structural variation in anaplasma marginale Msp2 efficiently generates immune escape variants

    Science.gov (United States)

    Antigenic variation allows microbial pathogens to evade immune clearance and establish persistent infection. Anaplasma marginale utilizes gene conversion of a repertoire of silent msp2 alleles into a single active expression site to encode unique Msp2 variants. As the genomic complement of msp2 alle...

  4. Proteomic profiling of SupT1 cells reveal modulation of host proteins by HIV-1 Nef variants.

    Directory of Open Access Journals (Sweden)

    Reshu Saxena

    Full Text Available Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1 through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01 in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1, VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 (55AAAAAAA61 and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein

  5. Optical generation of a spatially variant two-dimensional lattice structure by using a phase only spatial light modulator

    CERN Document Server

    Kumar, Manish

    2016-01-01

    We propose a simple and straightforward method to generate a spatially variant lattice structures by optical interference lithography method. Using this method, it is possible to independently vary the orientation and period of the two-dimensional lattice. The method consists of two steps which are: numerical synthesis of corresponding phase mask by employing a two-dimensional integrated gradient calculations and experimental implementation of synthesized phase mask by making use of a phase only spatial light modulator in an optical 4f Fourier filtering setup. As a working example, we provide the experimental fabrication of a spatially variant square lattice structure which has the possibility to guide a Gaussian beam through a 90{\\deg} bend by photonic crystal self-collimation phenomena. The method is digitally reconfigurable, is completely scalable and could be extended to other kind of lattices as well.

  6. Optical generation of a spatially variant two-dimensional lattice structure by using a phase only spatial light modulator

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manish, E-mail: manishk@physics.iitd.ac.in; Joseph, Joby, E-mail: joby@physics.iitd.ac.in [Photonics Research Laboratory, Department of Physics, Indian Institute of Technology Delhi, New Delhi 110016 (India)

    2014-08-04

    We propose a simple and straightforward method to generate spatially variant lattice structures by optical interference lithography method. Using this method, it is possible to independently vary the orientation and period of the two-dimensional lattice. The method consists of two steps which are: numerical synthesis of corresponding phase mask by employing a two-dimensional integrated gradient calculations and experimental implementation of synthesized phase mask by making use of a phase only spatial light modulator in an optical 4f Fourier filtering setup. As a working example, we provide the experimental fabrication of a spatially variant square lattice structure which has the possibility to guide a Gaussian beam through a 90° bend by photonic crystal self-collimation phenomena. The method is digitally reconfigurable, is completely scalable, and could be extended to other kind of lattices as well.

  7. Association of AKT1 gene variants and protein expression in both schizophrenia and bipolar disorder.

    Science.gov (United States)

    Karege, F; Perroud, N; Schürhoff, F; Méary, A; Marillier, G; Burkhardt, S; Ballmann, E; Fernandez, R; Jamain, S; Leboyer, M; La Harpe, R; Malafosse, A

    2010-07-01

    The AKT1 gene has been associated with the genetic aetiology of schizophrenia. Following the overlap model of bipolar disorder and schizophrenia, we aimed to investigate AKT1 genetic variants and protein expression in both diseases. A total of 679 subjects with European ancestry were included: 384 with schizophrenia, 130 with bipolar disorder and 165 controls. Six single nucleotide polymorphisms (SNPs) were investigated for association with the diseases using single- and multi-locus analyses. AKT1 and AKT2 protein levels were measured in post-mortem brain tissues from ante-mortem diagnosed schizophrenia (n = 30) and bipolar disorder subjects (n = 12) and matched controls. The analysis identified a significant global distortion in schizophrenia (P = 0.0026) and a weak association in bipolar disorder (P = 0.046). A sliding window procedure showed a five-SNP haplotype (TCGAG) to be associated with schizophrenia (P = 1.22 x 10(-4)) and bipolar disorder (P = 0.0041) and a four-SNP haplotype (TCGA) with the combined sample (1.73 x 10(-5)). On the basis of selected genotypes, a significant difference in protein expression emerged between subjects (P gene in both schizophrenia and bipolar disorder, support the role of AKT1 in the genetics of both disorders and add support to the view that there is some genetic overlap between them.

  8. Molecular modeling and molecular dynamic simulation of the effects of variants in the TGFBR2 kinase domain as a paradigm for interpretation of variants obtained by next generation sequencing.

    Science.gov (United States)

    Zimmermann, Michael T; Urrutia, Raul; Oliver, Gavin R; Blackburn, Patrick R; Cousin, Margot A; Bozeck, Nicole J; Klee, Eric W

    2017-01-01

    Variants in the TGFBR2 kinase domain cause several human diseases and can increase propensity for cancer. The widespread application of next generation sequencing within the setting of Individualized Medicine (IM) is increasing the rate at which TGFBR2 kinase domain variants are being identified. However, their clinical relevance is often uncertain. Consequently, we sought to evaluate the use of molecular modeling and molecular dynamics (MD) simulations for assessing the potential impact of variants within this domain. We documented the structural differences revealed by these models across 57 variants using independent MD simulations for each. Our simulations revealed various mechanisms by which variants may lead to functional alteration; some are revealed energetically, while others structurally or dynamically. We found that the ATP binding site and activation loop dynamics may be affected by variants at positions throughout the structure. This prediction cannot be made from the linear sequence alone. We present our structure-based analyses alongside those obtained using several commonly used genomics-based predictive algorithms. We believe the further mechanistic information revealed by molecular modeling will be useful in guiding the examination of clinically observed variants throughout the exome, as well as those likely to be discovered in the near future by clinical tests leveraging next-generation sequencing through IM efforts.

  9. Phage display selection of tight specific binding variants from a hyperthermostable Sso7d scaffold protein library.

    Science.gov (United States)

    Zhao, Ning; Schmitt, Margaret A; Fisk, John D

    2016-04-01

    Antibodies, the quintessential biological recognition molecules, are not ideal for many applications because of their large size, complex modifications, and thermal and chemical instability. Identifying alternative scaffolds that may be evolved into tight, specific binding molecules with improved physical properties is of increasing interest, particularly for biomedical applications in resource-limited environments. Hyperthermophilic organisms, such as Sulfolobus solfataricus, are an attractive source of highly stable proteins that may serve as starting points for alternative molecular recognition scaffolds. We describe the first application of phage display to identify binding proteins based on the S. solfataricus protein Sso7d scaffold. Sso7d is a small cysteine-free DNA-binding protein (approximately 7 kDa, 63 amino acids), with a melting temperature of nearly 100 °C. Tight-binding Sso7d variants were selected for a diverse set of protein targets from a 10(10) member library, demonstrating the versatility of the scaffold. These Sso7d variants are able to discriminate among closely related human, bovine and rabbit serum albumins. Equilibrium dissociation constants in the nanomolar to low micromolar range were measured via competitive ELISA. Importantly, the Sso7d variants continue to bind their targets in the absence of the phage context. Furthermore, phage-displayed Sso7d variants retain their binding affinity after exposure to temperatures up to 70 °C. Taken together, our results suggest that the Sso7d scaffold will be a complementary addition to the range of non-antibody scaffold proteins that may be utilized in phage display. Variants of hyperthermostable binding proteins have potential applications in diagnostics and therapeutics for environments with extreme conditions of storage and deployment.

  10. Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

    Science.gov (United States)

    Fisher, Kevin E.; Zhang, Linsheng; Wang, Jason; Smith, Geoffrey H.; Newman, Scott; Schneider, Thomas M.; Pillai, Rathi N.; Kudchadkar, Ragini R.; Owonikoko, Taofeek K.; Ramalingam, Suresh S.; Lawson, David H.; Delman, Keith A.; El-Rayes, Bassel F.; Wilson, Malania M.; Sullivan, H. Clifford; Morrison, Annie S.; Balci, Serdar; Adsay, N. Volkan; Gal, Anthony A.; Sica, Gabriel L.; Saxe, Debra F.; Mann, Karen P.; Hill, Charles E.; Khuri, Fadlo R.; Rossi, Michael R.

    2017-01-01

    We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated ≥500× coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at ≥5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n = 27), deletions (n = 5), insertions (n = 3), and multinucleotide variants (n = 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines. PMID:26801070

  11. Generation of transgenic Wuzhishan miniature pigs expressing monomeric red fluorescent protein by somatic cell nuclear transfer.

    Science.gov (United States)

    Lu, Yue; Kang, Jin-Dan; Li, Suo; Wang, Wei; Jin, Jun-Xue; Hong, Yu; Cui, Cheng-du; Yan, Chang-Guo; Yin, Xi-Jun

    2013-08-01

    Red fluorescent protein and its variants enable researchers to study gene expression, localization, and protein-protein interactions in vitro in real-time. Fluorophores with higher wavelengths are usually preferred since they efficiently penetrate tissues and produce less toxic emissions. A recently developed fluorescent protein marker, monomeric red fluorescent protein (mRFP1), is particularly useful because of its rapid maturation and minimal interference with green fluorescent protein (GFP) and GFP-derived markers. We generated a pCX-mRFP1-pgk-neoR construct and evaluated the ability of mRFP1 to function as a fluorescent marker in transgenic Wuzhishan miniature pigs. Transgenic embryos were generated by somatic cell nuclear transfer (SCNT) of nuclei isolated from ear fibroblasts expressing mRFP1. Embryos generated by SCNT developed into blastocysts in vitro (11.65%; 31/266). Thereafter, a total of 685 transgenic embryos were transferred into the oviducts of three recipients, two of which became pregnant. Of these, one recipient had six aborted fetuses, whereas the other recipient gave birth to four offspring. All offspring expressed the pCX-mRFP1-pgk-neoR gene as shown by PCR and fluorescence in situ hybridization analysis. The transgenic pigs expressed mRFP1 in all organs and tissues at high levels. These results demonstrate that Wuzhishan miniature pigs can express mRFP1. To conclude, this transgenic animal represents an excellent model with widespread applications in medicine and agriculture.

  12. Disparity map generation from illumination variant stereo images using efficient hierarchical dynamic programming.

    Science.gov (United States)

    Borisagar, Viral H; Zaveri, Mukesh A

    2014-01-01

    A novel hierarchical stereo matching algorithm is presented which gives disparity map as output from illumination variant stereo pair. Illumination difference between two stereo images can lead to undesirable output. Stereo image pair often experience illumination variations due to many factors like real and practical situation, spatially and temporally separated camera positions, environmental illumination fluctuation, and the change in the strength or position of the light sources. Window matching and dynamic programming techniques are employed for disparity map estimation. Good quality disparity map is obtained with the optimized path. Homomorphic filtering is used as a preprocessing step to lessen illumination variation between the stereo images. Anisotropic diffusion is used to refine disparity map to give high quality disparity map as a final output. The robust performance of the proposed approach is suitable for real life circumstances where there will be always illumination variation between the images. The matching is carried out in a sequence of images representing the same scene, however in different resolutions. The hierarchical approach adopted decreases the computation time of the stereo matching problem. This algorithm can be helpful in applications like robot navigation, extraction of information from aerial surveys, 3D scene reconstruction, and military and security applications. Similarity measure SAD is often sensitive to illumination variation. It produces unacceptable disparity map results for illumination variant left and right images. Experimental results show that our proposed algorithm produces quality disparity maps for both wide range of illumination variant and invariant stereo image pair.

  13. Disparity Map Generation from Illumination Variant Stereo Images Using Efficient Hierarchical Dynamic Programming

    Directory of Open Access Journals (Sweden)

    Viral H. Borisagar

    2014-01-01

    Full Text Available A novel hierarchical stereo matching algorithm is presented which gives disparity map as output from illumination variant stereo pair. Illumination difference between two stereo images can lead to undesirable output. Stereo image pair often experience illumination variations due to many factors like real and practical situation, spatially and temporally separated camera positions, environmental illumination fluctuation, and the change in the strength or position of the light sources. Window matching and dynamic programming techniques are employed for disparity map estimation. Good quality disparity map is obtained with the optimized path. Homomorphic filtering is used as a preprocessing step to lessen illumination variation between the stereo images. Anisotropic diffusion is used to refine disparity map to give high quality disparity map as a final output. The robust performance of the proposed approach is suitable for real life circumstances where there will be always illumination variation between the images. The matching is carried out in a sequence of images representing the same scene, however in different resolutions. The hierarchical approach adopted decreases the computation time of the stereo matching problem. This algorithm can be helpful in applications like robot navigation, extraction of information from aerial surveys, 3D scene reconstruction, and military and security applications. Similarity measure SAD is often sensitive to illumination variation. It produces unacceptable disparity map results for illumination variant left and right images. Experimental results show that our proposed algorithm produces quality disparity maps for both wide range of illumination variant and invariant stereo image pair.

  14. Interaction of mumps virus V protein variants with STAT1-STAT2 heterodimer: experimental and theoretical studies

    Directory of Open Access Journals (Sweden)

    Reyes-Leyva Julio

    2010-10-01

    Full Text Available Abstract Background Mumps virus V protein has the ability to inhibit the interferon-mediated antiviral response by inducing degradation of STAT proteins. Two virus variants purified from Urabe AM9 mumps virus vaccine differ in their replication and transcription efficiency in cells primed with interferon. Virus susceptibility to IFN was associated with insertion of a non-coded glycine at position 156 in the V protein (VGly of one virus variant, whereas resistance to IFN was associated with preservation of wild-type phenotype in the V protein (VWT of the other variant. Results VWT and VGly variants of mumps virus were cloned and sequenced from Urabe AM9 vaccine strain. VGly differs from VWT protein because it possesses an amino acid change Gln103Pro (Pro103 and the Gly156 insertion. The effect of V protein variants on components of the interferon-stimulated gene factor 3 (ISGF3, STAT1 and STAT2 proteins were experimentally tested in cervical carcinoma cell lines. Expression of VWT protein decreased STAT1 phosphorylation, whereas VGly had no inhibitory effect on either STAT1 or STAT2 phosphorylation. For theoretical analysis of the interaction between V proteins and STAT proteins, 3D structural models of VWT and VGly were predicted by comparing with simian virus 5 (SV5 V protein structure in complex with STAT1-STAT2 heterodimer. In silico analysis showed that VWT-STAT1-STAT2 complex occurs through the V protein Trp-motif (W174, W178, W189 and Glu95 residue close to the Arg409 and Lys415 of the nuclear localization signal (NLS of STAT2, leaving exposed STAT1 Lys residues (K85, K87, K296, K413, K525, K679, K685, which are susceptible to proteasome degradation. In contrast, the interaction between VGly and STAT1-STAT2 heterodimer occurs in a region far from the NLS of STAT2 without blocking of Lys residues in both STAT1 and STAT2. Conclusions Our results suggest that VWT protein of Urabe AM9 strain of mumps virus may be more efficient than VGly to

  15. Effects of breed and casein genetic variants on protein profile in milk from Swedish Red, Danish Holstein, and Danish Jersey

    DEFF Research Database (Denmark)

    Gustavsson, Frida; Buitenhuis, Albert Johannes; Johansson, M

    2014-01-01

    In selecting cows for higher milk yields and milk quality, it is important to understand how these traits are affected by the bovine genome. The major milk proteins exhibit genetic polymorphism and these genetic variants can serve as markers for milk composition, milk production traits, and techn......, for example, the processing of cheese...

  16. Hydrolysis of industrial substrates by an extremely stable thermolysin-like protease variant obtained by protein engineering

    NARCIS (Netherlands)

    Van den Burg, B; de Kreij, A; Winkel, C; Venema, G

    Various industrially used protein substrates were hydrolysed by a recently constructed, thermally stable, thermolysin-like protease variant (Boilysin; Van den Burg et al., Proc. Natl. Acad. Sci. 95: 2056-2060) and three industrial protease preparations. Hydrolysates were analysed by measuring the

  17. Prevalence of the prion protein gene E211K variant in U.S. cattle

    Directory of Open Access Journals (Sweden)

    Chase Chad C

    2008-07-01

    Full Text Available Abstract Background In 2006, an atypical U.S. case of bovine spongiform encephalopathy (BSE was discovered in Alabama and later reported to be polymorphic for glutamate (E and lysine (K codons at position 211 in the bovine prion protein gene (Prnp coding sequence. A bovine E211K mutation is important because it is analogous to the most common pathogenic mutation in humans (E200K which causes hereditary Creutzfeldt – Jakob disease, an autosomal dominant form of prion disease. The present report describes a high-throughput matrix-associated laser desorption/ionization-time-of-flight mass spectrometry assay for scoring the Prnp E211K variant and its use to determine an upper limit for the K211 allele frequency in U.S. cattle. Results The K211 allele was not detected in 6062 cattle, including those from five commercial beef processing plants (3892 carcasses and 2170 registered cattle from 42 breeds. Multiple nearby polymorphisms in Prnp coding sequence of 1456 diverse purebred cattle (42 breeds did not interfere with scoring E211 or K211 alleles. Based on these results, the upper bounds for prevalence of the E211K variant was estimated to be extremely low, less than 1 in 2000 cattle (Bayesian analysis based on 95% quantile of the posterior distribution with a uniform prior. Conclusion No groups or breeds of U.S. cattle are presently known to harbor the Prnp K211 allele. Because a carrier was not detected, the number of additional atypical BSE cases with K211 will also be vanishingly low.

  18. Toxic response caused by a misfolding variant of the mitochondrial protein short-chain acyl-CoA dehydrogenase

    DEFF Research Database (Denmark)

    Schmidt, Stinne P; Corydon, Thomas J; Pedersen, Christina B;

    2011-01-01

    the disease-associated misfolding variant of SCAD protein, p.Arg107Cys, disturbs mitochondrial function. METHODS: We have developed a cell model system, stably expressing either the SCAD wild-type protein or the misfolding SCAD variant protein, p.Arg107Cys (c.319 C > T). The model system was used......BACKGROUND: Variations in the gene ACADS, encoding the mitochondrial protein short-chain acyl CoA-dehydrogenase (SCAD), have been observed in individuals with clinical symptoms. The phenotype of SCAD deficiency (SCADD) is very heterogeneous, ranging from asymptomatic to severe, without a clear...... for investigation of SCAD with respect to expression, degree of misfolding, and enzymatic SCAD activity. Furthermore, cell proliferation and expression of selected stress response genes were investigated as well as proteomic analysis of mitochondria-enriched extracts in order to study the consequences of p.Arg107...

  19. Perturbation of myelin basic protein (Mbp) splice variant expression in developing rat cerebellum following perinatal exposure to methylmercury.

    Science.gov (United States)

    Padhi, Bhaja K; Pelletier, Guillaume

    2012-09-18

    Myelin sheaths surrounding axons are essential for saltatory conduction of nerve impulse in the central nervous system. A major protein constituent of myelin sheaths is produced by the myelin basic protein (Mbp) gene, whose expression in oligodendrocytes is conserved across vertebrates. In rat, five Mbp splice variants resulting from alternative splicing of exons 2, 5 and/or 6 are characterized. We developed a PCR-based strategy to quantify individual Mbp splice variants and characterized a sixth Mbp splice variant lacking only exon 5. This newly identified splice variant is predominantly expressed in developing rat brain and has orthologs in mouse and human. Many neurotoxic chemicals can perturb myelination and Mbp gene expression. Regulation of Mbp gene expression at the post-transcriptional level was assessed following perinatal exposure to neurotoxic methylmercury (2 mg/kg b.w./day). Similar reductions in total and individual Mbp splice variant mRNA levels suggest that methylmercury-induced perturbation in Mbp gene expression occurred as a consequence of decreased oligodendrocyte cell population in absence of a significant impact on its post-transcriptional regulation.

  20. Rabbit Hemorrhagic Disease Virus Variant Recombinant VP60 Protein Induces Protective Immunogenicity.

    Science.gov (United States)

    Yang, Dong-Kun; Kim, Ha-Hyun; Nah, Jin-Ju; Song, Jae-Young

    2015-11-01

    Rabbit hemorrhagic disease virus (RHDV) is highly contagious and often causes fatal disease that affects both wild and domestic rabbits of the species Oryctolagus cuniculus. A highly pathogenic RHDV variant (RHDVa) has been circulation in the Korean rabbit population since 2007 and has a devastating effect on the rabbit industry in Korea. A highly pathogenic RHDVa was isolated from naturally infected rabbits, and the gene encoding the VP60 protein was cloned into a baculovirus transfer vector and expressed in insect cells. The hemagglutination titer of the Sf-9 cell lysate infected with recombinant VP60 baculovirus was 131,072 units/50 μl and of the supernatant 4,096 units/50 μl. Guinea pigs immunized twice intramuscularly with a trial inactivated RHDVa vaccine containing recombinant VP60 contained 2,152 hemagglutination inhibition (HI) geometric mean titers. The 8-week-old white rabbits inoculated with one vaccine dose were challenged with a lethal RHDVa 21 days later and showed 100% survival rates. The recombinant VP60 protein expressed in a baculovirus system induced high HI titers in guinea pigs and rendered complete protection, which led to the development of a novel inactivated RHDVa vaccine.

  1. The Histone Deacetylase Complex 1 Protein of Arabidopsis Has the Capacity to Interact with Multiple Proteins Including Histone 3-Binding Proteins and Histone 1 Variants.

    Science.gov (United States)

    Perrella, Giorgio; Carr, Craig; Asensi-Fabado, Maria A; Donald, Naomi A; Páldi, Katalin; Hannah, Matthew A; Amtmann, Anna

    2016-05-01

    Intrinsically disordered proteins can adopt multiple conformations, thereby enabling interaction with a wide variety of partners. They often serve as hubs in protein interaction networks. We have previously shown that the Histone Deacetylase Complex 1 (HDC1) protein from Arabidopsis (Arabidopsis thaliana) interacts with histone deacetylases and quantitatively determines histone acetylation levels, transcriptional activity, and several phenotypes, including abscisic acid sensitivity during germination, vegetative growth rate, and flowering time. HDC1-type proteins are ubiquitous in plants, but they contain no known structural or functional domains. Here, we explored the protein interaction spectrum of HDC1 using a quantitative bimolecular fluorescence complementation assay in tobacco (Nicotiana benthamiana) epidermal cells. In addition to binding histone deacetylases, HDC1 directly interacted with histone H3-binding proteins and corepressor-associated proteins but not with H3 or the corepressors themselves. Surprisingly, HDC1 also was able to interact with variants of the linker histone H1. Truncation of HDC1 to the ancestral core sequence narrowed the spectrum of interactions and of phenotypic outputs but maintained binding to a H3-binding protein and to H1. Thus, HDC1 provides a potential link between H1 and histone-modifying complexes.

  2. A novel adaptive method for the analysis of next-generation sequencing data to detect complex trait associations with rare variants due to gene main effects and interactions.

    Directory of Open Access Journals (Sweden)

    Dajiang J Liu

    2010-10-01

    Full Text Available There is solid evidence that rare variants contribute to complex disease etiology. Next-generation sequencing technologies make it possible to uncover rare variants within candidate genes, exomes, and genomes. Working in a novel framework, the kernel-based adaptive cluster (KBAC was developed to perform powerful gene/locus based rare variant association testing. The KBAC combines variant classification and association testing in a coherent framework. Covariates can also be incorporated in the analysis to control for potential confounders including age, sex, and population substructure. To evaluate the power of KBAC: 1 variant data was simulated using rigorous population genetic models for both Europeans and Africans, with parameters estimated from sequence data, and 2 phenotypes were generated using models motivated by complex diseases including breast cancer and Hirschsprung's disease. It is demonstrated that the KBAC has superior power compared to other rare variant analysis methods, such as the combined multivariate and collapsing and weight sum statistic. In the presence of variant misclassification and gene interaction, association testing using KBAC is particularly advantageous. The KBAC method was also applied to test for associations, using sequence data from the Dallas Heart Study, between energy metabolism traits and rare variants in ANGPTL 3,4,5 and 6 genes. A number of novel associations were identified, including the associations of high density lipoprotein and very low density lipoprotein with ANGPTL4. The KBAC method is implemented in a user-friendly R package.

  3. Effect of Variants of Penicillin-Binding Protein 2 on Cephalosporin and Carbapenem Susceptibilities in Neisseria gonorrhoeae.

    Science.gov (United States)

    Bharat, Amrita; Demczuk, Walter; Martin, Irene; Mulvey, Michael R

    2015-08-01

    To characterize the relationship between penicillin-binding protein 2 (PBP2/penA) and susceptibility to extended-spectrum cephalosporins (ESCs) and carbapenem antibiotics, we compared 17 PBP2 variants in Neisseria gonorrhoeae. Nonmosaic and mosaic variants of PBP2 caused decreased susceptibility to ESCs and, to a lesser extent, to carbapenems. An A501P substitution in mosaic XXXIV_A501P conferred decreased susceptibility to ESCs but restored carbapenem susceptibility to wild-type levels. These results could aid the molecular surveillance of antimicrobial resistance to these agents.

  4. Strategies for purifying variants of human rhinovirus 14 2C protein.

    Science.gov (United States)

    Sára, Tomáš; Konrat, Robert; Skern, Tim

    2014-03-01

    The positive strand RNA genome of picornaviruses, including human rhinovirus (HRV), poliovirus (PV) and foot-and-mouth disease virus, is translated immediately into a polyprotein that is cleaved by virally encoded proteinases into 10-13 mature proteins. These include the four proteins required to assemble the viral particle as well as 3D(pol) (the viral RNA polymerase) and 2C, an ATPase and putative helicase. 2C is a protein which is responsible, together with 2B and 3A, for anchoring the replication complexes to membranous structures in the infected cell on which RNA replication takes place. Additionally, expression of 2C and its precursor 2BC in mammalian cells leads to vesicle formation observed in infected cells. 2C is encoded by all picornaviruses; nevertheless, its exact role in viral replication remains unclear. A contributing factor is the absence of structural data for this hydrophobic protein the generation of which has been hampered by an inability to produce soluble and stable material. Here, we compare 2C from several genera and show that the 2C protein has considerable heterogeneity. Using protein structure meta-analysis, we developed models of HRV14 2C that should be useful for mutational analysis. Based on these analyses, we expressed and purified two domains of HRV14 2C using three different protocols and examined the folding by thermal denaturation or (1)H NMR. Both domains were concentrated sufficiently to allow crystal screens or NMR pilot experiments to be performed. This work provides a platform to explore 2C proteins from all picornaviral genera to generate candidates for structural analysis.

  5. A reference data set of 5.4 million phased human variants validated by genetic inheritance from sequencing a three-generation 17-member pedigree

    Science.gov (United States)

    Eberle, Michael A.; Fritzilas, Epameinondas; Krusche, Peter; Källberg, Morten; Moore, Benjamin L.; Bekritsky, Mitchell A.; Iqbal, Zamin; Chuang, Han-Yu; Humphray, Sean J.; Halpern, Aaron L.; Kruglyak, Semyon; Margulies, Elliott H.; McVean, Gil; Bentley, David R.

    2017-01-01

    Improvement of variant calling in next-generation sequence data requires a comprehensive, genome-wide catalog of high-confidence variants called in a set of genomes for use as a benchmark. We generated deep, whole-genome sequence data of 17 individuals in a three-generation pedigree and called variants in each genome using a range of currently available algorithms. We used haplotype transmission information to create a phased “Platinum” variant catalog of 4.7 million single-nucleotide variants (SNVs) plus 0.7 million small (1–50 bp) insertions and deletions (indels) that are consistent with the pattern of inheritance in the parents and 11 children of this pedigree. Platinum genotypes are highly concordant with the current catalog of the National Institute of Standards and Technology for both SNVs (>99.99%) and indels (99.92%) and add a validated truth catalog that has 26% more SNVs and 45% more indels. Analysis of 334,652 SNVs that were consistent between informatics pipelines yet inconsistent with haplotype transmission (“nonplatinum”) revealed that the majority of these variants are de novo and cell-line mutations or reside within previously unidentified duplications and deletions. The reference materials from this study are a resource for objective assessment of the accuracy of variant calls throughout genomes. PMID:27903644

  6. Protein adsorption on ion exchange resins and monoclonal antibody charge variant modulation.

    Science.gov (United States)

    Guélat, Bertrand; Khalaf, Rushd; Lattuada, Marco; Costioli, Matteo; Morbidelli, Massimo

    2016-05-20

    A novel multicomponent adsorption equilibrium model for proteins on ion-exchange resins is developed on a statistical thermodynamic basis including surface coverage effects and protein-resin and protein-protein interactions. The resulting model exhibits a general competitive Langmuirian behavior and was applied to the study and optimization of the separation of monoclonal antibody charge variants on two strong cation exchangers. The model accounts explicitly for the effect of both pH and salt concentration, and its parameters can be determined in diluted conditions, that is, through physically sound assumptions, all model parameters can be obtained using solely experiments in diluted conditions, and be used to make predictions in overloaded conditions. The parameterization of the model and optimization of the separation is based on a two-step approach. First, gradient experiments in diluted conditions are undertaken in order to determine the model parameters. Based on these experiments and on information about the proteins of interest and the stationary phase used, all the model parameters can be estimated. Second, using the parameterized model, an initial Pareto optimization is undertaken where overloaded operating conditions are investigated. Experiments from this Pareto set are then used to refine the estimation of the model parameters. A second Pareto optimization can then be undertaken, this time with the refined parameters. This can be repeated until a satisfactory set of model parameters is found. This iterative approach is shown to be extremely efficient and to provide large amounts of knowledge based on only a few experiments. It is shown that due to the strong physical foundation of the model and the very low number of adjustable parameters, the number of iterations is expected to be at most two or three. Furthermore, the model based tool is improved as more experimental knowledge is provided, allowing for better estimations of the chromatographic

  7. Advances in generating functional diversity for directed protein evolution.

    Science.gov (United States)

    Shivange, Amol V; Marienhagen, Jan; Mundhada, Hemanshu; Schenk, Alexander; Schwaneberg, Ulrich

    2009-02-01

    Despite advances in screening technologies, only a very small fraction of theoretical protein sequence can be sampled in directed evolution experiments. At the current state of random mutagenesis technologies mutation frequencies have often been adjusted to values that cause a limited number of amino acid changes (often one to four amino acid changes per protein). For harvesting the power of directed evolution algorithms it is therefore important that generated mutant libraries are rich in diversity and enriched in active population. Insufficient knowledge about protein traits, mutational robustness of protein folds and technological limitations in diversity generating methods are main challenges for managing the complexity of protein sequence space. This review covers computational and experimental advances for high quality mutant library generation that have been achieved in the past two years.

  8. PPG: online generation of protein pictures and animations.

    Science.gov (United States)

    Binisti, Cédric; Salim, Ahmed Ali; Tufféry, Pierre

    2005-07-01

    The protein picture generator (PPG) is an online service to generate pictures of a protein structure. Its design was conceived as an answer to the need expressed by a part of the community to have some means to produce simply complex pictures to insert in publications or in presentations. PPG can produce static or animated pictures. It can be accessed at http://bioserv.rpbs.jussieu.fr/cgi-bin/PPG.

  9. Gene transfer and expression of enhanced green fluorescent protein in variant HT-29c cells

    Institute of Scientific and Technical Information of China (English)

    Min Wang; Lars Boenicke; Bradley D. Howard; Ilka Vogel; Hoiger Kalthoff

    2003-01-01

    AIM: To study the expression of enhanced green fluorescent protein (EGFP) gene in retrovirally transduced variant HT29 cells.METHODS: The retroviral vector prkat EGFP/neo was constructed and transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (selected from HT-29 cells) were transduced by a retroviral vector encoding the GEFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transduced with the EGFP bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter (FACS) analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransduced and transduced cells in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo.RESULTS: After transduced, HT-29c cells displayed a stable and long-term EGFP expression under the nonselective conditionsin vitro. After cells were successively cultured to passage 50 in vitro, EGFP expression was still at a high level. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransduced parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed.CONCLUSION: An EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo.These cells with EGFP are a valuable tool forin vivo research of tumor metastatic spread.

  10. No evidence that protein truncating variants in BRIP1 are associated with breast cancer risk

    DEFF Research Database (Denmark)

    Easton, Douglas F; Lesueur, Fabienne; Decker, Brennan

    2016-01-01

    in BRIP1 are associated with an increased risk of breast cancer. These studies have led to inclusion of BRIP1 on targeted sequencing panels for breast cancer risk prediction. METHODS: We evaluated a truncating variant, p.Arg798Ter (rs137852986), and 10 missense variants of BRIP1, in 48 144 cases and 43...

  11. No evidence that protein truncating variants in BRIP1 are associated with breast cancer risk: implications for gene panel testing

    Science.gov (United States)

    Easton, Douglas F; Lesueur, Fabienne; Decker, Brennan; Michailidou, Kyriaki; Li, Jun; Allen, Jamie; Luccarini, Craig; Pooley, Karen A; Shah, Mitul; Bolla, Manjeet K; Wang, Qin; Dennis, Joe; Ahmad, Jamil; Thompson, Ella R; Damiola, Francesca; Pertesi, Maroulio; Voegele, Catherine; Mebirouk, Noura; Robinot, Nivonirina; Durand, Geoffroy; Forey, Nathalie; Luben, Robert N; Ahmed, Shahana; Aittomäki, Kristiina; Anton-Culver, Hoda; Arndt, Volker; Baynes, Caroline; Beckman, Matthias W; Benitez, Javier; Van Den Berg, David; Blot, William J; Bogdanova, Natalia V; Bojesen, Stig E; Brenner, Hermann; Chang-Claude, Jenny; Chia, Kee Seng; Choi, Ji-Yeob; Conroy, Don M; Cox, Angela; Cross, Simon S; Czene, Kamila; Darabi, Hatef; Devilee, Peter; Eriksson, Mikael; Fasching, Peter A; Figueroa, Jonine; Flyger, Henrik; Fostira, Florentia; García-Closas, Montserrat; Giles, Graham G; Glendon, Gord; González-Neira, Anna; Guénel, Pascal; Haiman, Christopher A; Hall, Per; Hart, Steven N; Hartman, Mikael; Hooning, Maartje J; Hsiung, Chia-Ni; Ito, Hidemi; Jakubowska, Anna; James, Paul A; John, Esther M; Johnson, Nichola; Jones, Michael; Kabisch, Maria; Kang, Daehee; Kosma, Veli-Matti; Kristensen, Vessela; Lambrechts, Diether; Li, Na; Lindblom, Annika; Long, Jirong; Lophatananon, Artitaya; Lubinski, Jan; Mannermaa, Arto; Manoukian, Siranoush; Margolin, Sara; Matsuo, Keitaro; Meindl, Alfons; Mitchell, Gillian; Muir, Kenneth; Nevelsteen, Ines; van den Ouweland, Ans; Peterlongo, Paolo; Phuah, Sze Yee; Pylkäs, Katri; Rowley, Simone M; Sangrajrang, Suleeporn; Schmutzler, Rita K; Shen, Chen-Yang; Shu, Xiao-Ou; Southey, Melissa C; Surowy, Harald; Swerdlow, Anthony; Teo, Soo H; Tollenaar, Rob A E M; Tomlinson, Ian; Torres, Diana; Truong, Thérèse; Vachon, Celine; Verhoef, Senno; Wong-Brown, Michelle; Zheng, Wei; Zheng, Ying; Nevanlinna, Heli; Scott, Rodney J; Andrulis, Irene L; Wu, Anna H; Hopper, John L; Couch, Fergus J; Winqvist, Robert; Burwinkel, Barbara; Sawyer, Elinor J; Schmidt, Marjanka K; Rudolph, Anja; Dörk, Thilo; Brauch, Hiltrud; Hamann, Ute; Neuhausen, Susan L; Milne, Roger L; Fletcher, Olivia; Pharoah, Paul D P; Campbell, Ian G; Dunning, Alison M; Le Calvez-Kelm, Florence; Goldgar, David E; Tavtigian, Sean V; Chenevix-Trench, Georgia

    2016-01-01

    Background BRCA1 interacting protein C-terminal helicase 1 (BRIP1) is one of the Fanconi Anaemia Complementation (FANC) group family of DNA repair proteins. Biallelic mutations in BRIP1 are responsible for FANC group J, and previous studies have also suggested that rare protein truncating variants in BRIP1 are associated with an increased risk of breast cancer. These studies have led to inclusion of BRIP1 on targeted sequencing panels for breast cancer risk prediction. Methods We evaluated a truncating variant, p.Arg798Ter (rs137852986), and 10 missense variants of BRIP1, in 48 144 cases and 43 607 controls of European origin, drawn from 41 studies participating in the Breast Cancer Association Consortium (BCAC). Additionally, we sequenced the coding regions of BRIP1 in 13 213 cases and 5242 controls from the UK, 1313 cases and 1123 controls from three population-based studies as part of the Breast Cancer Family Registry, and 1853 familial cases and 2001 controls from Australia. Results The rare truncating allele of rs137852986 was observed in 23 cases and 18 controls in Europeans in BCAC (OR 1.09, 95% CI 0.58 to 2.03, p=0.79). Truncating variants were found in the sequencing studies in 34 cases (0.21%) and 19 controls (0.23%) (combined OR 0.90, 95% CI 0.48 to 1.70, p=0.75). Conclusions These results suggest that truncating variants in BRIP1, and in particular p.Arg798Ter, are not associated with a substantial increase in breast cancer risk. Such observations have important implications for the reporting of results from breast cancer screening panels. PMID:26921362

  12. Molecular characterization of a CpTRIM35-like protein and its splice variants from whitespotted bamboo shark (Chiloscyllium plagiosum)

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xinshang, E-mail: sanmaosound@163.com; Zhao, Heng, E-mail: hengzhao2000@gmail.com; Chen, Yeyu, E-mail: cyyleaf@126.com; Luo, Huiying, E-mail: luohuiying@caas.cn; Yao, Bin, E-mail: binyao@caas.cn

    2014-10-24

    Highlights: • A TRIM gene and three splice variants were firstly cloned from elasmobranch fish. • The genes were constitutively expressed with high levels in spleen and kidney. • The gene products were distributed in cytoplasm alone or cytoplasm and nucleus. • As E3 ubiquitin ligases, the proteins differed in immune responses to challenges. - Abstract: The tripartite motif (TRIM) proteins play important roles in a broad range of biological processes, including apoptosis, cell proliferation and innate immunity response. In this study, a TRIM gene and its three splice variants were cloned from an elasmobranch fish—whitespotted bamboo shark (Chiloscyllium plagiosum Bennett). Phylogenetic analysis indicated that the gene was closely related to TRIM35 homologs, thus termed CpTRIM35-like. Deduced CpTRIM35 has a RBCC-PRY/SPRY structure typical of TRIM proteins, and its splice variants (CpTRIM35-1–3) have different truncations at the C-terminus. The gene products were constitutively expressed in adult sharks with the highest levels in spleen and kidney. The different subcellular locations, upregulation upon LPS and poly I:C stimulation, and significant E3 ubiquitin ligase activities suggested their different roles in immune responses as an E3 ubiquitin ligase. This is the first TRIM protein ever characterized in elasmobranch fish.

  13. A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens.

    Directory of Open Access Journals (Sweden)

    Emmanuel Y Dotsey

    Full Text Available In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001 with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV.

  14. Characterization of functional methylomes by next-generation capture sequencing identifies novel disease-associated variants.

    Science.gov (United States)

    Allum, Fiona; Shao, Xiaojian; Guénard, Frédéric; Simon, Marie-Michelle; Busche, Stephan; Caron, Maxime; Lambourne, John; Lessard, Julie; Tandre, Karolina; Hedman, Åsa K; Kwan, Tony; Ge, Bing; Rönnblom, Lars; McCarthy, Mark I; Deloukas, Panos; Richmond, Todd; Burgess, Daniel; Spector, Timothy D; Tchernof, André; Marceau, Simon; Lathrop, Mark; Vohl, Marie-Claude; Pastinen, Tomi; Grundberg, Elin

    2015-05-29

    Most genome-wide methylation studies (EWAS) of multifactorial disease traits use targeted arrays or enrichment methodologies preferentially covering CpG-dense regions, to characterize sufficiently large samples. To overcome this limitation, we present here a new customizable, cost-effective approach, methylC-capture sequencing (MCC-Seq), for sequencing functional methylomes, while simultaneously providing genetic variation information. To illustrate MCC-Seq, we use whole-genome bisulfite sequencing on adipose tissue (AT) samples and public databases to design AT-specific panels. We establish its efficiency for high-density interrogation of methylome variability by systematic comparisons with other approaches and demonstrate its applicability by identifying novel methylation variation within enhancers strongly correlated to plasma triglyceride and HDL-cholesterol, including at CD36. Our more comprehensive AT panel assesses tissue methylation and genotypes in parallel at ∼4 and ∼3 M sites, respectively. Our study demonstrates that MCC-Seq provides comparable accuracy to alternative approaches but enables more efficient cataloguing of functional and disease-relevant epigenetic and genetic variants for large-scale EWAS.

  15. G23D: Online tool for mapping and visualization of genomic variants on 3D protein structures

    OpenAIRE

    Solomon, Oz; Kunik, Vered; Simon, Amos; Kol, Nitzan; Barel, Ortal; Lev, Atar; Amariglio, Ninette; Somech, Raz; Rechavi, Gidi; Eyal, Eran

    2016-01-01

    Background Evaluation of the possible implications of genomic variants is an increasingly important task in the current high throughput sequencing era. Structural information however is still not routinely exploited during this evaluation process. The main reasons can be attributed to the partial structural coverage of the human proteome and the lack of tools which conveniently convert genomic positions, which are the frequent output of genomic pipelines, to proteins and structure coordinates...

  16. Generation and propagation of radical reactions on proteins

    DEFF Research Database (Denmark)

    Hawkins, C L; Davies, Michael Jonathan

    2001-01-01

    The oxidation of proteins by free radicals is thought to play a major role in many oxidative processes within cells and is implicated in a number of human diseases as well as ageing. This review summarises information on the formation of radicals on peptides and proteins and how radical damage may...... be propagated and transferred within protein structures. The emphasis of this article is primarily on the deleterious actions of radicals generated on proteins, and their mechanisms of action, rather than on enzymatic systems where radicals are deliberately formed as transient intermediates. The final section...

  17. A scallop IGF binding protein gene: molecular characterization and association of variants with growth traits.

    Directory of Open Access Journals (Sweden)

    Liying Feng

    Full Text Available BACKGROUND: Scallops represent economically important aquaculture shellfish. The identification of genes and genetic variants related to scallop growth could benefit high-yielding scallop breeding. The insulin-like growth factor (IGF system is essential for growth and development, with IGF binding proteins (IGFBPs serving as the major regulators of IGF actions. Although an effect of IGF on growth was detected in bivalve, IGFBP has not been reported, and members of the IGF system have not been characterized in scallop. RESULTS: We cloned and characterized an IGFBP (PyIGFBP gene from the aquaculture bivalve species, Yesso scallop (Patinopecten yessoensis, Jay, 1857. Its full-length cDNA sequence was 1,445 bp, with an open reading frame of 378 bp, encoding 125 amino acids, and its genomic sequence was 10,193 bp, consisting of three exons and two introns. The amino acid sequence exhibited the characteristics of IGFBPs, including multiple cysteine residues and relatively conserved motifs in the N-terminal and C-terminal domains. Expression analysis indicated that PyIGFBP was expressed in all the tissues and developmental stages examined, with a significantly higher level in the mantle than in other tissues and a significantly higher level in gastrulae and trochophore larvae than in other stages. Furthermore, three single nucleotide polymorphisms (SNPs were identified in this gene. SNP c.1054A>G was significantly associated with both shell and soft body traits in two populations, with the highest trait values in GG type scallops and lowest in AG type ones. CONCLUSION: We cloned and characterized an IGFBP gene in a bivalve, and this report also represents the first characterizing an IGF system gene in scallops. A SNP associated with scallop growth for both the shell and soft body was identified in this gene. In addition to providing a candidate marker for scallop breeding, our results also suggest the role of PyIGFBP in scallop growth.

  18. Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population

    DEFF Research Database (Denmark)

    Berquist, Brian R; Singh, Dharmendra Kumar; Fan, Jinshui

    2010-01-01

    ) and two frequent (R194W and R399Q) amino acid population variants had little or no effect on XRCC1 protein stability or the interactions with POLbeta, PARP-1, LIG3alpha, PCNA or DNA. One common population variant (R280H) had no pronounced effect on the interactions with POLbeta, PARP-1, LIG3alpha and PCNA...

  19. MET receptor variant R970C favors calpain-dependent generation of a fragment promoting epithelial cell scattering.

    Science.gov (United States)

    Montagne, Rémi; Baranzelli, Anne; Muharram, Ghaffar; Catherine, Leroy; Lesaffre, Marie; Vinchent, Audrey; Kherrouche, Zoulika; Werkmeister, Elisabeth; Cortot, Alexis B; Tulasne, David

    2017-01-04

    The receptor tyrosine kinase MET and its ligand, the hepatocyte growth factor, are essential to embryonic development, whereas deregulation of MET signaling is associated with tumorigenesis leading to various cancers, including lung carcinoma. Mutations in the MET kinase domain lead to constitutive kinase activity and are associated with tumorigenesis. In lung cancer, however, some mutations are found in the juxtamembrane domain, and their functional consequences are unknown. Because the juxtamembrane domain of MET is targeted by several proteolytic cleavages, involved in its degradation during cell death or under steady-state conditions, we evaluated the influence of these mutations on the MET proteolytic cleavages. In stably transfected epithelial cells expressing MET, the juxtamembrane mutations R970C, P991S, and T992I were found not to modify the known caspase or presenilin-dependent regulated intramembrane proteolysis. Yet when overexpressed, the R970C variant caused generation of an as yet undescribed 45-kDa fragment (p45 MET). This fragment was found in the confluent lung cancer cell line NCI-H1437 carrying the R970C mutation and at a lesser extent in cell lines expressing WT MET, suggesting that R970C mutation favors this cleavage. Generation of p45 MET required the activity of the calpain proteases, confirming the involvement of proteolysis. Ectopic expression of reconstituted p45 MET in epithelial cell lines favored cell scattering and invasion indicating active role of this fragment in HGF/SF induced responses. Hence, although the juxtamembrane mutations of MET do not affect its known proteolytic cleavages, the R970C MET variant favors calpain dependent proteolytic cleavage in lung cancer cells.

  20. Generation of protein-derived redox cofactors by posttranslational modification.

    Science.gov (United States)

    Davidson, Victor L

    2011-01-01

    Redox enzymes which catalyze the oxidation and reduction of substrates are ubiquitous in nature. These enzymes typically possess exogenous cofactors to allow them to perform catalytic functions which cannot be accomplished using only amino acid residues. It is now evident that nature also employs an alternative strategy of generating catalytic and redox-active sites in proteins by posttranslational modification of amino acid residues. This review describes the structures and functions of several of these protein-derived cofactors and the diverse mechanisms of posttranslational modification through which they are generated.

  1. A TIR domain receptor-associated protein (TIRAP) variant SNP (rs8177374) confers protection against premature birth.

    Science.gov (United States)

    Karody, V R; Le, M; Nelson, S; Meskin, K; Klemm, S; Simpson, P; Hines, R; Sampath, V

    2013-05-01

    To investigate whether single nucleotide polymorphisms (SNPs) in genes encoding the Toll-like receptor (TLR) signaling pathway modulate susceptibility to preterm birth (PTB). Prospective case-control study examining the contribution of nine TLR SNPs to PTB (<37 weeks) and PTB <32 weeks. Genotyping was done on neonatal blood using a multiplexed single-base extension assay. Chi-square test, Fischer's exact test and classification trees were used for data analysis. Preterm infants (n=177) were more likely to be African American (P=0.02), and were more likely to be born to mothers who smoked (P=0.007), had pregnancy-induced hypertension (PIH; P=0.002) and placental abruption (P=0.0004) when compared with term infants (n=146). The TLR2, TLR4, TLR5, TLR9, nuclear factor-kappa B1 (NFκB1), NFκBIA and IRAK1 variants were not associated with PTB whereas the TIR domain receptor-associated protein (TIRAP) variant was more prevalent in term infants when compared with preterm infants born <32 weeks (P=0.004). PTB <32 weeks was more prevalent in infants without the TIRAP variant whose mothers had PIH and did not smoke (P=0.001). Presence of the TIRAP variant protected against PTB <32 weeks (P=0.015) in Caucasian infants. In our study, a TLR pathway adapter variant (TIRAP (rs8177374)) protected against PTB<32 weeks, supporting our hypothesis that genetic variation in the innate immune signaling pathway contributes to altered risk of PTB.

  2. A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates.

    Directory of Open Access Journals (Sweden)

    Jihong Li

    2008-05-01

    Full Text Available Clostridium perfringens is a major cause of food poisoning (FP in developed countries. C. perfringens isolates usually induce the gastrointestinal symptoms of this FP by producing an enterotoxin that is encoded by a chromosomal (cpe gene. Those typical FP strains also produce spores that are extremely resistant to food preservation approaches such as heating and chemical preservatives. This resistance favors their survival and subsequent germination in improperly cooked, prepared, or stored foods. The current study identified a novel alpha/beta-type small acid soluble protein, now named Ssp4, and showed that sporulating cultures of FP isolates producing resistant spores consistently express a variant Ssp4 with an Asp substitution at residue 36. In contrast, Gly was detected at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Studies with isogenic mutants and complementing strains demonstrated the importance of the Asp 36 Ssp4 variant for the exceptional heat and sodium nitrite resistance of spores made by most FP strains carrying a chromosomal cpe gene. Electrophoretic mobility shift assays and DNA binding studies showed that Ssp4 variants with an Asp at residue 36 bind more efficiently and tightly to DNA than do Ssp4 variants with Gly at residue 36. Besides suggesting one possible mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene, these findings may facilitate eventual development of targeted strategies to increase killing of the resistant spores in foods. They also provide the first indication that SASP variants can be important contributors to intra-species (and perhaps inter-species variations in bacterial spore resistance phenotypes. Finally, Ssp4 may contribute to spore resistance properties throughout the genus Clostridium since ssp4 genes also exist in the genomes of other clostridial species.

  3. A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates.

    Science.gov (United States)

    Li, Jihong; McClane, Bruce A

    2008-05-02

    Clostridium perfringens is a major cause of food poisoning (FP) in developed countries. C. perfringens isolates usually induce the gastrointestinal symptoms of this FP by producing an enterotoxin that is encoded by a chromosomal (cpe) gene. Those typical FP strains also produce spores that are extremely resistant to food preservation approaches such as heating and chemical preservatives. This resistance favors their survival and subsequent germination in improperly cooked, prepared, or stored foods. The current study identified a novel alpha/beta-type small acid soluble protein, now named Ssp4, and showed that sporulating cultures of FP isolates producing resistant spores consistently express a variant Ssp4 with an Asp substitution at residue 36. In contrast, Gly was detected at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Studies with isogenic mutants and complementing strains demonstrated the importance of the Asp 36 Ssp4 variant for the exceptional heat and sodium nitrite resistance of spores made by most FP strains carrying a chromosomal cpe gene. Electrophoretic mobility shift assays and DNA binding studies showed that Ssp4 variants with an Asp at residue 36 bind more efficiently and tightly to DNA than do Ssp4 variants with Gly at residue 36. Besides suggesting one possible mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene, these findings may facilitate eventual development of targeted strategies to increase killing of the resistant spores in foods. They also provide the first indication that SASP variants can be important contributors to intra-species (and perhaps inter-species) variations in bacterial spore resistance phenotypes. Finally, Ssp4 may contribute to spore resistance properties throughout the genus Clostridium since ssp4 genes also exist in the genomes of other clostridial species.

  4. PeSV-Fisher: identification of somatic and non-somatic structural variants using next generation sequencing data.

    Directory of Open Access Journals (Sweden)

    Geòrgia Escaramís

    Full Text Available UNLABELLED: Next-generation sequencing technologies expedited research to develop efficient computational tools for the identification of structural variants (SVs and their use to study human diseases. As deeper data is obtained, the existence of higher complexity SVs in some genomes becomes more evident, but the detection and definition of most of these complex rearrangements is still in its infancy. The full characterization of SVs is a key aspect for discovering their biological implications. Here we present a pipeline (PeSV-Fisher for the detection of deletions, gains, intra- and inter-chromosomal translocations, and inversions, at very reasonable computational costs. We further provide comprehensive information on co-localization of SVs in the genome, a crucial aspect for studying their biological consequences. The algorithm uses a combination of methods based on paired-reads and read-depth strategies. PeSV-Fisher has been designed with the aim to facilitate identification of somatic variation, and, as such, it is capable of analysing two or more samples simultaneously, producing a list of non-shared variants between samples. We tested PeSV-Fisher on available sequencing data, and compared its behaviour to that of frequently deployed tools (BreakDancer and VariationHunter. We have also tested this algorithm on our own sequencing data, obtained from a tumour and a normal blood sample of a patient with chronic lymphocytic leukaemia, on which we have also validated the results by targeted re-sequencing of different kinds of predictions. This allowed us to determine confidence parameters that influence the reliability of breakpoint predictions. AVAILABILITY: PeSV-Fisher is available at http://gd.crg.eu/tools.

  5. Hydrogen Exchange Mass Spectrometry of Related Proteins with Divergent Sequences: A Comparative Study of HIV-1 Nef Allelic Variants

    Science.gov (United States)

    Wales, Thomas E.; Poe, Jerrod A.; Emert-Sedlak, Lori; Morgan, Christopher R.; Smithgall, Thomas E.; Engen, John R.

    2016-06-01

    Hydrogen exchange mass spectrometry can be used to compare the conformation and dynamics of proteins that are similar in tertiary structure. If relative deuterium levels are measured, differences in sequence, deuterium forward- and back-exchange, peptide retention time, and protease digestion patterns all complicate the data analysis. We illustrate what can be learned from such data sets by analyzing five variants (Consensus G2E, SF2, NL4-3, ELI, and LTNP4) of the HIV-1 Nef protein, both alone and when bound to the human Hck SH3 domain. Regions with similar sequence could be compared between variants. Although much of the hydrogen exchange features were preserved across the five proteins, the kinetics of Nef binding to Hck SH3 were not the same. These observations may be related to biological function, particularly for ELI Nef where we also observed an impaired ability to downregulate CD4 surface presentation. The data illustrate some of the caveats that must be considered for comparison experiments and provide a framework for investigations of other protein relatives, families, and superfamilies with HX MS.

  6. A Klothoβ variant mediates protein stability and associates with colon transit in irritable bowel syndrome with diarrhea.

    Science.gov (United States)

    Wong, Banny S; Camilleri, Michael; Carlson, Paula J; Guicciardi, Maria E; Burton, Duane; McKinzie, Sanna; Rao, Archana S; Zinsmeister, Alan R; Gores, Gregory J

    2011-06-01

    Bile acid (BA) malabsorption of moderate severity is reported in 32% of patients with chronic unexplained diarrhea, including diarrhea-predominant irritable bowel syndrome (IBS-D). We hypothesized that variants of genes regulating hepatic BA synthesis play a role in IBS-D. In 435 IBS and 279 healthy subjects, we tested individual associations of 15 common single nucleotide polymorphisms (SNPs) from 7 genes critical to BA homeostasis with symptom-based subgroups using dominant genetic models. In a subset of 238 participants, we tested association with colonic transit. SNP-SNP interactions were investigated based on known protein interactions in BA homeostasis. The function of SNP rs17618244 in Klothoβ (KLB) was evaluated using a protein stability assay in HEK293 cells. SNP rs17618244 (Arg728Gln in KLB) is associated with colonic transit at 24 hours. G allele (Arg728) compared with A allele (Gln728) is associated with accelerated colonic transit (P=.0007) in the overall cohort; this association was restricted to IBS-D (P=.0018). Interaction tests of KLB rs17618244 with 3 nonsynonymous SNPs of fibroblast growth factor receptor 4 (FGFR4) revealed that rs1966265 (Val10Ile) and rs351855 (Gly388Arg) modulate rs1768244's association with colonic transit in IBS-D (P=.0025 and P=.0023, respectively). KLB Arg728 significantly reduced protein stability compared with KLB Gln728, demonstrating KLB rs17618244's functional significance. No significant associations with symptom-based subgroups of IBS were detected. A functional KLB gene variant mediating protein stability associates with colonic transit in IBS-D. This association is modulated by 2 genetic variants in FGFR4. The FGF19-FGFR4-KLB pathway links regulation of BA synthesis to colonic transit in IBS-D. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

  7. An ATP and oxalate generating variant tricarboxylic acid cycle counters aluminum toxicity in Pseudomonas fluorescens.

    Directory of Open Access Journals (Sweden)

    Ranji Singh

    Full Text Available Although the tricarboxylic acid (TCA cycle is essential in almost all aerobic organisms, its precise modulation and integration in global cellular metabolism is not fully understood. Here, we report on an alternative TCA cycle uniquely aimed at generating ATP and oxalate, two metabolites critical for the survival of Pseudomonas fluorescens. The upregulation of isocitrate lyase (ICL and acylating glyoxylate dehydrogenase (AGODH led to the enhanced synthesis of oxalate, a dicarboxylic acid involved in the immobilization of aluminum (Al. The increased activity of succinyl-CoA synthetase (SCS and oxalate CoA-transferase (OCT in the Al-stressed cells afforded an effective route to ATP synthesis from oxalyl-CoA via substrate level phosphorylation. This modified TCA cycle with diminished efficacy in NADH production and decreased CO(2-evolving capacity, orchestrates the synthesis of oxalate, NADPH, and ATP, ingredients pivotal to the survival of P. fluorescens in an Al environment. The channeling of succinyl-CoA towards ATP formation may be an important function of the TCA cycle during anaerobiosis, Fe starvation and O(2-limited conditions.

  8. Lead Generation and Optimization Based on Protein-Ligand Complementarity

    Directory of Open Access Journals (Sweden)

    Koji Ogata

    2010-06-01

    Full Text Available This work proposes a computational procedure for structure-based lead generation and optimization, which relies on the complementarity of the protein-ligand interactions. This procedure takes as input the known structure of a protein-ligand complex. Retaining the positions of the ligand heavy atoms in the protein binding site it designs structurally similar compounds considering all possible combinations of atomic species (N, C, O, CH3, NH,etc. Compounds are ranked based on a score which incorporates energetic contributions evaluated using molecular mechanics force fields. This procedure was used to design new inhibitor molecules for three serine/threonine protein kinases (p38 MAP kinase, p42 MAP kinase (ERK2, and c-Jun N-terminal kinase 3 (JNK3. For each enzyme, the calculations produce a set of potential inhibitors whose scores are in agreement with IC50 data and Ki values. Furthermore, the native ligands for each protein target, scored within the five top-ranking compounds predicted by our method, one of the top-ranking compounds predicted to inhibit JNK3 was synthesized and his inhibitory activity confirmed against ATP hydrolysis. Our computational procedure is therefore deemed to be a useful tool for generating chemically diverse molecules active against known target proteins.

  9. Staphylococcus aureus isolates encode variant staphylococcal enterotoxin B proteins that are diverse in superantigenicity and lethality.

    Directory of Open Access Journals (Sweden)

    Petra L Kohler

    Full Text Available Staphylococcus aureus produces superantigens (SAgs that bind and cross-link T cells and APCs, leading to activation and proliferation of immune cells. SAgs bind to variable regions of the β-chains of T cell receptors (Vβ-TCRs, and each SAg binds a unique subset of Vβ-TCRs. This binding leads to massive cytokine production and can result in toxic shock syndrome (TSS. The most abundantly produced staphylococcal SAgs and the most common causes of staphylococcal TSS are TSS toxin-1 (TSST-1, and staphylococcal enterotoxins B and C (SEB and SEC, respectively. There are several characterized variants of humans SECs, designated SEC1-4, but only one variant of SEB has been described. Sequencing the seb genes from over 20 S. aureus isolates show there are at least five different alleles of seb, encoding forms of SEB with predicted amino acid substitutions outside of the predicted immune-cell binding regions of the SAgs. Examination of purified, variant SEBs indicates that these amino acid substitutions cause differences in proliferation of rabbit splenocytes in vitro. Additionally, the SEBs varied in lethality in a rabbit model of TSS. The SEBs were diverse in their abilities to cause proliferation of human peripheral blood mononuclear cells, and differed in their activation of subsets of T cells. A soluble, high-affinity Vβ-TCR, designed to neutralize the previously characterized variant of SEB (SEB1, was able to neutralize the variant SEBs, indicating that this high-affinity peptide may be useful in treating a variety of SEB-mediated illnesses.

  10. BRCA1 gene variant p.P142H associated with male breast cancer: a two-generation genealogic study and literature review.

    Science.gov (United States)

    Spinelli, Claudio; Strambi, Silvia; Piccini, Lorenzo; Rossi, Leonardo; Aretini, Paolo; Caligo, Adelaide

    2015-12-01

    Breast cancer occurs rarely in male patient. BRCA1 gene mutation seems to be related to male breast cancer, but its role is not clearly defined. We have identified in a male patient affected by breast cancer the BRCA1 gene variant p.P142H. We performed a literature research using the keywords "male breast cancer", "male breast cancer mutations" and "BRCA" and we reviewed the cases. We found ew other studies regarding BRCA1 variant p.P142H, about female subjects. At the moment, BRCA1 gene variant p.P142H is not certainly classified as neutral or deleterious. Genetic testing for BRCA1 and BRCA2 and PALB2 mutation gene has been performed on our patient. Segregation analysis for this p.P142H BRCA1 variant has been extended to the second generation of the family. Genetic tests revealed a clear inheritance regarding the BRCA1 gene p. P142H variant. Of the eight patients with this specific genetic mutation, four presented breast cancer (bilateral in one case), two female and two male. None of the subjects in the family without the BRCA1 gene variant p. P142H presented breast cancer or other BRCA1 gene mutation-related cancers. Our analysis suggests that the BRCA1 gene variant p.P142H mutation is related with male breast cancer. Starting from these data, it can be inferred that more studies on MBC and its relation with the BRCA1 gene mutation P142H variant must be undertaken to improve prognostic and therapeutic strategies.

  11. A new strategy for enhancing imputation quality of rare variants from next-generation sequencing data via combining SNP and exome chip data

    NARCIS (Netherlands)

    Y.J. Kim (Young Jin); J. Lee (Juyoung); B.-J. Kim (Bong-Jo); T. Park (Taesung); G.R. Abecasis (Gonçalo); M. Almeida (Marcio); D. Altshuler (David); J.L. Asimit (Jennifer L.); G. Atzmon (Gil); M. Barber (Mathew); A. Barzilai (Ari); N.L. Beer (Nicola L.); G.I. Bell (Graeme I.); J. Below (Jennifer); T. Blackwell (Tom); J. Blangero (John); M. Boehnke (Michael); D.W. Bowden (Donald W.); N.P. Burtt (Noël); J.C. Chambers (John); H. Chen (Han); P. Chen (Ping); P.S. Chines (Peter); S. Choi (Sungkyoung); C. Churchhouse (Claire); P. Cingolani (Pablo); B.K. Cornes (Belinda); N.J. Cox (Nancy); A.G. Day-Williams (Aaron); A. Duggirala (Aparna); J. Dupuis (Josée); T. Dyer (Thomas); S. Feng (Shuang); J. Fernandez-Tajes (Juan); T. Ferreira (Teresa); T.E. Fingerlin (Tasha E.); J. Flannick (Jason); J.C. Florez (Jose); P. Fontanillas (Pierre); T.M. Frayling (Timothy); C. Fuchsberger (Christian); E. Gamazon (Eric); K. Gaulton (Kyle); S. Ghosh (Saurabh); B. Glaser (Benjamin); A.L. Gloyn (Anna); R.L. Grossman (Robert L.); J. Grundstad (Jason); C. Hanis (Craig); A. Heath (Allison); H. Highland (Heather); M. Horikoshi (Momoko); I.-S. Huh (Ik-Soo); J.R. Huyghe (Jeroen R.); M.K. Ikram (Kamran); K.A. Jablonski (Kathleen); Y. Jun (Yang); N. Kato (Norihiro); J. Kim (Jayoun); Y.J. Kim (Young Jin); B.-J. Kim (Bong-Jo); J. Lee (Juyoung); C.R. King (C. Ryan); J.S. Kooner (Jaspal S.); M.-S. Kwon (Min-Seok); H.K. Im (Hae Kyung); M. Laakso (Markku); K.K.-Y. Lam (Kevin Koi-Yau); J. Lee (Jaehoon); S. Lee (Selyeong); S. Lee (Sungyoung); D.M. Lehman (Donna M.); H. Li (Heng); C.M. Lindgren (Cecilia); X. Liu (Xuanyao); O.E. Livne (Oren E.); A.E. Locke (Adam E.); A. Mahajan (Anubha); J.B. Maller (Julian B.); A.K. Manning (Alisa K.); T.J. Maxwell (Taylor J.); A. Mazoure (Alexander); M.I. McCarthy (Mark); J.B. Meigs (James B.); B. Min (Byungju); K.L. Mohlke (Karen); A.P. Morris (Andrew); S. Musani (Solomon); Y. Nagai (Yoshihiko); M.C.Y. Ng (Maggie C.Y.); D. Nicolae (Dan); S. Oh (Sohee); N.D. Palmer (Nicholette); T. Park (Taesung); T.I. Pollin (Toni I.); I. Prokopenko (Inga); D. Reich (David); M.A. Rivas (Manuel); L.J. Scott (Laura); M. Seielstad (Mark); Y.S. Cho (Yoon Shin); X. Sim (Xueling); R. Sladek (Rob); P. Smith (Philip); I. Tachmazidou (Ioanna); E.S. Tai (Shyong); Y.Y. Teo (Yik Ying); T.M. Teslovich (Tanya M.); J. Torres (Jason); V. Trubetskoy (Vasily); S.M. Willems (Sara); A.L. Williams (Amy L.); J.G. Wilson (James); S. Wiltshire (Steven); S. Won (Sungho); A.R. Wood (Andrew); W. Xu (Wang); J. Yoon (Joon); M. Zawistowski (Matthew); E. Zeggini (Eleftheria); W. Zhang (Weihua); S. Zöllner (Sebastian)

    2015-01-01

    textabstractBackground: Rare variants have gathered increasing attention as a possible alternative source of missing heritability. Since next generation sequencing technology is not yet cost-effective for large-scale genomic studies, a widely used alternative approach is imputation. However, the imp

  12. Community proteogenomics highlights microbial strain-variant protein expression within activated sludge performing enhanced biological phosphorus removal.

    Energy Technology Data Exchange (ETDEWEB)

    Wilmes, P [University of California, Berkeley; Andersson, Anders F. [University of California, Berkeley; Lefsrud, Mark G [McGill University, Montreal, Quebec; Wexler, Margaret [University of East Anglia, Norwich, United Kingdom; Shah, Manesh B [ORNL; Zhang, B [Vanderbilt University; Hettich, Robert {Bob} L [ORNL; Bond, P. L. [University of Queensland, The, Brisbane, Queensland, Australia; Verberkmoes, Nathan C [ORNL; Banfield, Jillian F. [University of California, Berkeley

    2008-01-01

    Enhanced biological phosphorus removal (EBPR) selects for polyphosphate accumulating organisms to achieve phosphate removal from wastewater. We used highresolution community proteomics to identify key metabolic pathways in "Candidatus Accumulibacter phosphatis"-mediated EBPR and to evaluate the contributions of co- 5 existing strains within the dominant population. Results highlight the importance of denitrification, fatty acid cycling and the glyoxylate bypass in EBPR. Despite overall strong similarity in protein profiles under anaerobic and aerobic conditions, fatty acid degradation proteins were more abundant during the anaerobic phase. By comprehensive genome-wide alignment of orthologous proteins, we uncovered strong 10 functional partitioning for enzyme variants involved in both core-metabolism and EBPR-specific pathways among the dominant strains. These findings emphasize the importance of genetic diversity in maintaining the stable performance of EBPR systems and demonstrate the power of integrated cultivation-independent genomics and proteomics for analysis of complex biotechnological systems.

  13. Variant size- and glycoforms of the scavenger receptor cysteine-rich protein gp-340 with differential bacterial aggregation

    DEFF Research Database (Denmark)

    Eriksson, Christer; Frängsmyr, Lars; Danielsson Niemi, Liza

    2007-01-01

    Glycoprotein gp-340 aggregates bacteria in saliva as part of innate defence at mucosal surfaces. We have detected size- and glycoforms of gp-340 between human saliva samples (n = 7) and lung gp-340 from a proteinosis patient using antibodies and lectins in Western blots and ELISA measurements...... bands. Purified I to IV proteins all revealed a N-terminal sequence TGGWIP upon Edman degradation. Moreover, purified gp-340 from the seven donors and lung gp-340 shared N-glycans, sialylated Galbeta1-3GalNAc and (poly)lactosamine structures. However, the larger size gp-340 grouping II/III (n = 4....... Western blots of saliva samples, and of gp-340 purified, from the seven donors using a gp-340 specific antibody distinguished four gp-340 size variants, designated I to IV (n = 2,2,2 and 1). While saliva gp-340 variants I to III had single bands of increasing sizes, variant IV and lung gp-340 had double...

  14. Molecular mechanism of a green-shifted, pH-dependent red fluorescent protein mKate variant.

    Directory of Open Access Journals (Sweden)

    Qi Wang

    Full Text Available Fluorescent proteins that can switch between distinct colors have contributed significantly to modern biomedical imaging technologies and molecular cell biology. Here we report the identification and biochemical analysis of a green-shifted red fluorescent protein variant GmKate, produced by the introduction of two mutations into mKate. Although the mutations decrease the overall brightness of the protein, GmKate is subject to pH-dependent, reversible green-to-red color conversion. At physiological pH, GmKate absorbs blue light (445 nm and emits green fluorescence (525 nm. At pH above 9.0, GmKate absorbs 598 nm light and emits 646 nm, far-red fluorescence, similar to its sequence homolog mNeptune. Based on optical spectra and crystal structures of GmKate in its green and red states, the reversible color transition is attributed to the different protonation states of the cis-chromophore, an interpretation that was confirmed by quantum chemical calculations. Crystal structures reveal potential hydrogen bond networks around the chromophore that may facilitate the protonation switch, and indicate a molecular basis for the unusual bathochromic shift observed at high pH. This study provides mechanistic insights into the color tuning of mKate variants, which may aid the development of green-to-red color-convertible fluorescent sensors, and suggests GmKate as a prototype of genetically encoded pH sensors for biological studies.

  15. A generative, probabilistic model of local protein structure

    DEFF Research Database (Denmark)

    Boomsma, Wouter; Mardia, Kanti V.; Taylor, Charles C.;

    2008-01-01

    Despite significant progress in recent years, protein structure prediction maintains its status as one of the prime unsolved problems in computational biology. One of the key remaining challenges is an efficient probabilistic exploration of the structural space that correctly reflects the relative...... conformational stabilities. Here, we present a fully probabilistic, continuous model of local protein structure in atomic detail. The generative model makes efficient conformational sampling possible and provides a framework for the rigorous analysis of local sequence-structure correlations in the native state...

  16. Protein C system defects inflicted by the malaria parasite protein PfEMP1 can be overcome by a soluble EPCR variant

    DEFF Research Database (Denmark)

    Petersen, Jens E V; Bouwens, Eveline A M; Tamayo, Ibai;

    2015-01-01

    The Endothelial Protein C receptor (EPCR) is essential for the anticoagulant and cytoprotective functions of the Protein C (PC) system. Selected variants of the malaria parasite protein, Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) associated with severe malaria, including cerebral...... malaria, specifically target EPCR on vascular endothelial cells. Here, we examine the cellular response to PfEMP1 engagement to elucidate its role in malaria pathogenesis. Binding of the CIDRα1.1 domain of PfEMP1 to EPCR obstructed activated PC (APC) binding to EPCR and induced a loss of cellular EPCR...... not interfere with (A)PC binding to cellular EPCR. E86A-sEPCR used as a decoy to capture PfEMP1, permitted normal PC activation on endothelial cells, normal barrier protective effects of APC, and greatly reduced cytoadhesion of infected erythrocytes to brain endothelial cells. These data imply important...

  17. Divergence in the plasminogen-binding group a streptococcal M protein family: functional conservation of binding site and potential role for immune selection of variants.

    Science.gov (United States)

    Sanderson-Smith, Martina; Batzloff, Michael; Sriprakash, Kabada S; Dowton, Mark; Ranson, Marie; Walker, Mark J

    2006-02-10

    Group A streptococci (GAS) display receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M protein (PAM). Characterization of PAM genes from 12 GAS isolates showed significant variation within the plasminogen-binding repeat motifs (a1/a2) of this protein. To determine the impact of sequence variation on protein function, recombinant proteins representing five naturally occurring variants of PAM, together with a recombinant M1 protein, were expressed and purified. Equilibrium dissociation constants for the interaction of PAM variants with biotinylated Glu-plasminogen ranged from 1.58 to 4.99 nm. Effective concentrations of prototype PAM required for 50% inhibition of plasminogen binding to immobilized PAM variants ranged from 0.68 to 22.06 nm. These results suggest that although variation in the a1/a2 region of the PAM protein does affect the comparative affinity of PAM variants, the functional capacity to bind plasminogen is conserved. Additionally, a potential role for the a1 region of PAM in eliciting a protective immune response was investigated by using a mouse model for GAS infection. The a1 region of PAM was found to protect immunized mice challenged with a PAM-positive GAS strain. These data suggest a link between selective immune pressure against the plasminogen-binding repeats and the functional conservation of the binding domain in PAM variants.

  18. NRAS germline variant G138R and multiple rare somatic mutations on APC in colorectal cancer patients in Taiwan by next generation sequencing.

    Science.gov (United States)

    Chang, Pi-Yueh; Chen, Jinn-Shiun; Chang, Nai-Chung; Chang, Shih-Cheng; Wang, Mei-Chia; Tsai, Shu-Hui; Wen, Ying-Hao; Tsai, Wen-Sy; Chan, Err-Cheng; Lu, Jang-Jih

    2016-06-21

    Colorectal cancer (CRC) arises from mutations in a subset of genes. We investigated the germline and somatic mutation spectrum of patients with CRC in Taiwan by using the AmpliSeq Cancer Hotspot Panel V2. Fifty paired freshly frozen stage 0-IV CRC tumors and adjacent normal tissue were collected. Blood DNA from 20 healthy donors were used for comparison of germline mutations. Variants were identified using an ion-torrent personal genomic machine and subsequently confirmed by Sanger sequencing or pyrosequencing. Five nonsynonymous germline variants on 4 cancer susceptible genes, CDH1, APC, MLH1, and NRAS, were observed in 6 patients with CRC (12%). Among them, oncogene NRAS G138R variant was identified as having a predicted damaging effect on protein function, which has never been reported by other laboratories. CDH1 T340A variants were presented in 3 patients. The germline variants in the cancer patients differed completely from those found in asymptomatic controls. Furthermore, a total of 56 COSMIC and 21 novel somatic variants distributed in 20 genes were detected in 44 (88%) of the CRC samples. High inter- and intra-tumor heterogeneity levels were observed. Nine rare variants located in the β-catenin binding region of the APC gene were discovered, 7 of which could cause amino acid frameshift and might have a pathogenic effect. In conclusion, panel-based mutation detection by using a high-throughput sequencing platform can elucidate race-dependent cancer genomes. This approach facilitates identifying individuals at high risk and aiding the recognition of novel mutations as targets for drug development.

  19. Timing the generation of distinct retinal cells by homeobox proteins.

    Directory of Open Access Journals (Sweden)

    Sarah Decembrini

    2006-09-01

    Full Text Available The reason why different types of vertebrate nerve cells are generated in a particular sequence is still poorly understood. In the vertebrate retina, homeobox genes play a crucial role in establishing different cell identities. Here we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. By in situ expression analysis, we found that the three Xenopus homeobox genes Xotx5b, Xvsx1, and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b and bipolar cells (Xvsx1 and Xotx2. Furthermore, by in vivo lipofection of "sensors" in which green fluorescent protein translation is under control of the 3' untranslated region (UTR, we found that the 3' UTRs of Xotx5b, Xvsx1, and Xotx2 are sufficient to drive a spatiotemporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b and bipolar cells (Xvsx1 and Xotx2. The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequences, respectively. This is the first evidence to our knowledge that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities.

  20. Common exonic missense variants in the C2 domain of the human KIBRA protein modify lipid binding and cognitive performance.

    Science.gov (United States)

    Duning, K; Wennmann, D O; Bokemeyer, A; Reissner, C; Wersching, H; Thomas, C; Buschert, J; Guske, K; Franzke, V; Flöel, A; Lohmann, H; Knecht, S; Brand, S-M; Pöter, M; Rescher, U; Missler, M; Seelheim, P; Pröpper, C; Boeckers, T M; Makuch, L; Huganir, R; Weide, T; Brand, E; Pavenstädt, H; Kremerskothen, J

    2013-06-18

    The human KIBRA gene has been linked to human cognition through a lead intronic single-nucleotide polymorphism (SNP; rs17070145) that is associated with episodic memory performance and the risk to develop Alzheimer's disease. However, it remains unknown how this relates to the function of the KIBRA protein. Here, we identified two common missense SNPs (rs3822660G/T [M734I], rs3822659T/G [S735A]) in exon 15 of the human KIBRA gene to affect cognitive performance, and to be in almost complete linkage disequilibrium with rs17070145. The identified SNPs encode variants of the KIBRA C2 domain with distinct Ca(2+) dependent binding preferences for monophosphorylated phosphatidylinositols likely due to differences in the dynamics and folding of the lipid-binding pocket. Our results further implicate the KIBRA protein in higher brain function and provide direction to the cellular pathways involved.

  1. Common exonic missense variants in the C2 domain of the human KIBRA protein modify lipid binding and cognitive performance

    Science.gov (United States)

    Duning, K; Wennmann, D O; Bokemeyer, A; Reissner, C; Wersching, H; Thomas, C; Buschert, J; Guske, K; Franzke, V; Flöel, A; Lohmann, H; Knecht, S; Brand, S-M; Pöter, M; Rescher, U; Missler, M; Seelheim, P; Pröpper, C; Boeckers, T M; Makuch, L; Huganir, R; Weide, T; Brand, E; Pavenstädt, H; Kremerskothen, J

    2013-01-01

    The human KIBRA gene has been linked to human cognition through a lead intronic single-nucleotide polymorphism (SNP; rs17070145) that is associated with episodic memory performance and the risk to develop Alzheimer's disease. However, it remains unknown how this relates to the function of the KIBRA protein. Here, we identified two common missense SNPs (rs3822660G/T [M734I], rs3822659T/G [S735A]) in exon 15 of the human KIBRA gene to affect cognitive performance, and to be in almost complete linkage disequilibrium with rs17070145. The identified SNPs encode variants of the KIBRA C2 domain with distinct Ca2+ dependent binding preferences for monophosphorylated phosphatidylinositols likely due to differences in the dynamics and folding of the lipid-binding pocket. Our results further implicate the KIBRA protein in higher brain function and provide direction to the cellular pathways involved. PMID:23778582

  2. A group of Giardia lamblia variant-specific surface protein (VSP) genes with nearly identical 5' regions.

    Science.gov (United States)

    Yang, Y; Adam, R D

    1995-12-01

    The surfaces of Giardia lamblia trophozoites contain one of a set of variant-specific surface proteins. The genes encoding these proteins are highly conserved at the 3' terminus, but frequently demonstrate little similarity in the remainder of the coding region. This report describes a family of vsp genes highly similar to a repeat-containing vsp gene (vspC5) at the 5' coding and flanking regions, but which diverge abruptly from vspC5 in the first repeat and do not themselves contain full copies of the repeat. This observation suggests the possibility that recombination among different vsp genes may have played a role in development of the vsp gene repertoire.

  3. Cloning and identification of NS5ATP2 gene and its spliced variant transactivated by hepatitis C virus non-structural protein 5A

    Institute of Scientific and Technical Information of China (English)

    Qian Yang; Jun Cheng; Yan Liu; Yuan Hong; Jian-Jun Wang; Shu-Lin Zhang

    2004-01-01

    AIM: To clone, identify and study new NS5ATP2 gene and its spliced variant transactivated by hepatitis C virus nonstructural protein 5A.METHODS: On the basis of subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus, the coding sequence of new gene and its spliced variant were obtained by bioinformatics method. Polymerase chain reaction (PCR)was conducted to amplify NS5ATP2 gene.RESUJLTS: The coding sequence of a new gene and its spliced variant were cloned and identified successfully.CONCLUSION: A new gene has been recognized as the new target transactivated by HCV NS5A protein. These results brought some new clues for studying the biological functions of new genes and pathogenesis of the viral proteins.

  4. Expression of recombinant murine pregnancy-associated plasma protein-A (PAPP-A) and a novel variant (PAPP-Ai) with differential proteolytic activity

    DEFF Research Database (Denmark)

    Søe, Rikke; Overgaard, Michael Toft; Thomsen, Anni R

    2002-01-01

    Murine pregnancy-associated plasma protein-A (PAPP-A) cDNA encoding a 1545 amino-acid protein has been cloned. We have also identified and cloned cDNA that encodes a novel variant of PAPP-A, PAPP-Ai, carrying a 29-residue highly basic insert. The point of insertion corresponds to a junction between...

  5. Comparative Study on the Infectivity and Spore Surface Protein of Nosema bombycis and Its Morphological Variant Strain

    Institute of Scientific and Technical Information of China (English)

    HUANG Shao-kang; LU Xing-meng

    2005-01-01

    A new morphological variant strain of microsporidium was produced by infecting the mulberry looper, Hemerophila atrilineata [Phthonandria atrilineata], with Nosema bombycis successively for 24 times, and named 24Nbh. Comparative studies on morphology, infectivity and spore surface protein were conducted. 24Nbh was short and wide, and had a significant difference (P<0.01) over the Nb spores. The infectivity tests conducted on second instar silkworm larvae showed that IC50 of 24Nbh was 1.98× 104 spores mL-1 and of Nb was 1.72× 103 spores mL-1, thus indicating that the infectivity of Nb decreased 11.5 times after multiplying in mulberry looper for 24 times. The IC50 of spores from silkworm infected with 24 Nbh was 6.9 times less than Nb, showing that the infectivity of 24Nbh spores rejuvenated very fast when reinfected to silkworms, further more, the length and width of such spore was larger than 24Nbh (P<0.01) and smaller than Nb (P<0.05).The SDS-PAGE profiles of Nb and 24Nbh were generally the same, 4 distinct proteins of 12, 17, 30, 33 kDa were obtained with difference in quantity. When 120 μg of protein was applied for 2D-PAGE, five suspected different proteins with difference in quantity were observed. These results demonstrate that these differential proteins maybe associated with variation in infectivity of the spores.

  6. Accurate variant detection across non-amplified and whole genome amplified DNA using targeted next generation sequencing

    Directory of Open Access Journals (Sweden)

    ElSharawy Abdou

    2012-09-01

    Full Text Available Abstract Background Many hypothesis-driven genetic studies require the ability to comprehensively and efficiently target specific regions of the genome to detect sequence variations. Often, sample availability is limited requiring the use of whole genome amplification (WGA. We evaluated a high-throughput microdroplet-based PCR approach in combination with next generation sequencing (NGS to target 384 discrete exons from 373 genes involved in cancer. In our evaluation, we compared the performance of six non-amplified gDNA samples from two HapMap family trios. Three of these samples were also preamplified by WGA and evaluated. We tested sample pooling or multiplexing strategies at different stages of the tested targeted NGS (T-NGS workflow. Results The results demonstrated comparable sequence performance between non-amplified and preamplified samples and between different indexing strategies [sequence specificity of 66.0% ± 3.4%, uniformity (coverage at 0.2× of the mean of 85.6% ± 0.6%]. The average genotype concordance maintained across all the samples was 99.5% ± 0.4%, regardless of sample type or pooling strategy. We did not detect any errors in the Mendelian patterns of inheritance of genotypes between the parents and offspring within each trio. We also demonstrated the ability to detect minor allele frequencies within the pooled samples that conform to predicted models. Conclusion Our described PCR-based sample multiplex approach and the ability to use WGA material for NGS may enable researchers to perform deep resequencing studies and explore variants at very low frequencies and cost.

  7. Generation, Fractionation, and Characterization of Iron-Chelating Protein Hydrolysate from Palm Kernel Cake Proteins.

    Science.gov (United States)

    Zarei, Mohammad; Ghanbari, Rahele; Tajabadi, Naser; Abdul-Hamid, Azizah; Bakar, Fatimah Abu; Saari, Nazamid

    2016-02-01

    Palm kernel cake protein was hydrolyzed with different proteases namely papain, bromelain, subtilisin, flavourzyme, trypsin, chymotrypsin, and pepsin to generate different protein hydrolysates. Peptide content and iron-chelating activity of each hydrolysate were evaluated using O-phthaldialdehyde-based spectrophotometric method and ferrozine-based colorimetric assay, respectively. The results revealed a positive correlation between peptide contents and iron-chelating activities of the protein hydrolysates. Protein hydrolysate generated by papain exhibited the highest peptide content of 10.5 mM and highest iron-chelating activity of 64.8% compared with the other hydrolysates. Profiling of the papain-generated hydrolysate by reverse phase high performance liquid chromatography fractionation indicated a direct association between peptide content and iron-chelating activity in most of the fractions. Further fractionation using isoelectric focusing also revealed that protein hydrolysate with basic and neutral isoelectric point (pI) had the highest iron-chelating activity, although a few fractions in the acidic range also exhibited good metal chelating potential. After identification and synthesis of papain-generated peptides, GGIF and YLLLK showed among the highest iron-chelating activities of 56% and 53%, whereas their IC50 were 1.4 and 0.2 μM, respectively.

  8. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.

    Science.gov (United States)

    Kumar, Prateek; Henikoff, Steven; Ng, Pauline C

    2009-01-01

    The effect of genetic mutation on phenotype is of significant interest in genetics. The type of genetic mutation that causes a single amino acid substitution (AAS) in a protein sequence is called a non-synonymous single nucleotide polymorphism (nsSNP). An nsSNP could potentially affect the function of the protein, subsequently altering the carrier's phenotype. This protocol describes the use of the 'Sorting Tolerant From Intolerant' (SIFT) algorithm in predicting whether an AAS affects protein function. To assess the effect of a substitution, SIFT assumes that important positions in a protein sequence have been conserved throughout evolution and therefore substitutions at these positions may affect protein function. Thus, by using sequence homology, SIFT predicts the effects of all possible substitutions at each position in the protein sequence. The protocol typically takes 5-20 min, depending on the input. SIFT is available as an online tool (http://sift.jcvi.org).

  9. Fatty-acid binding protein 4 gene variants and childhood obesity: potential implications for insulin sensitivity and CRP levels

    Directory of Open Access Journals (Sweden)

    Bhattacharjee Rakesh

    2010-02-01

    Full Text Available Abstract Introduction Obesity increases the risk for insulin resistance and metabolic syndrome in both adults and children. FABP4 is a member of the intracellular lipid-binding protein family that is predominantly expressed in adipose tissue, and plays an important role in maintaining glucose and lipid homeostasis. The purpose of this study was to measure FABP4 plasma levels, assess FABP4 allelic variants, and explore potential associations with fasting glucose and insulin levels in young school-age children with and without obesity. Methods A total of 309 consecutive children ages 5-7 years were recruited. Children were divided based on BMI z score into Obese (OB; BMI z score >1.65 and non-obese (NOB. Fasting plasma glucose, lipids, insulin, hsCRP, and FABP4 levels were measured. HOMA was used as correlate of insulin sensitivity. Four SNPs of the human FABP4 gene (rs1051231, rs2303519, rs16909233 and rs1054135, corresponding to several critical regions of the encoding FABP4 gene sequence were genotyped. Results Compared to NOB, circulating FABP4 levels were increased in OB, as were LDL, hsCRP and HOMA. FABP4 levels correlated with BMI, and also contributed to the variance of HOMA and hsCRP, but not serum lipids. The frequency of rs1054135 allelic variant was increased in OB, and was associated with increased FABP4 levels, while the presence of rs16909233 variant allele, although similar in OB and NOB, was associated with increased HOMA values. Conclusions Childhood obesity is associated with higher FABP4 levels that may promote cardiometabolic risk. The presence of selective SNPs in the FABP4 gene may account for increased risk for insulin resistance or systemic inflammation in the context of obesity.

  10. Seed Dormancy in Arabidopsis Requires Self-Binding Ability of DOG1 Protein and the Presence of Multiple Isoforms Generated by Alternative Splicing.

    Directory of Open Access Journals (Sweden)

    Kazumi Nakabayashi

    2015-12-01

    Full Text Available The Arabidopsis protein DELAY OF GERMINATION 1 (DOG1 is a key regulator of seed dormancy, which is a life history trait that determines the timing of seedling emergence. The amount of DOG1 protein in freshly harvested seeds determines their dormancy level. DOG1 has been identified as a major dormancy QTL and variation in DOG1 transcript levels between accessions contributes to natural variation for seed dormancy. The DOG1 gene is alternatively spliced. Alternative splicing increases the transcriptome and proteome diversity in higher eukaryotes by producing transcripts that encode for proteins with altered or lost function. It can also generate tissue specific transcripts or affect mRNA stability. Here we suggest a different role for alternative splicing of the DOG1 gene. DOG1 produces five transcript variants encoding three protein isoforms. Transgenic dog1 mutant seeds expressing single DOG1 transcript variants from the endogenous DOG1 promoter did not complement because they were non-dormant and lacked DOG1 protein. However, transgenic plants overexpressing single DOG1 variants from the 35S promoter could accumulate protein and showed complementation. Simultaneous expression of two or more DOG1 transcript variants from the endogenous DOG1 promoter also led to increased dormancy levels and accumulation of DOG1 protein. This suggests that single isoforms are functional, but require the presence of additional isoforms to prevent protein degradation. Subsequently, we found that the DOG1 protein can bind to itself and that this binding is required for DOG1 function but not for protein accumulation. Natural variation for DOG1 binding efficiency was observed among Arabidopsis accessions and contributes to variation in seed dormancy.

  11. Surfactant Protein-D-Encoding Gene Variant Polymorphisms Are Linked to Respiratory Outcome in Premature Infants

    DEFF Research Database (Denmark)

    Sorensen, Grith Lykke; Dahl, Marianne; Tan, Qihua

    2014-01-01

    OBJECTIVE: Associations between the genetic variation within or downstream of the surfactant protein-D-encoding gene (SFTPD), which encodes the collectin surfactant protein-D (SP-D) and may lead to respiratory distress syndrome or bronchopulmonary dysplasia, recently were reported. Our aim was to...

  12. Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Bao, Baolong [Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE (United States); Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education (China); Wijeratne, Subhashinee S.K.; Rodriguez-Melendez, Rocio [Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE (United States); Zempleni, Janos, E-mail: jzempleni2@unl.edu [Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE (United States)

    2011-08-19

    Highlights: {yields} Unambiguous evidence is provided that methionine-58 serves as an in-frame alternative translation site for holocarboxylase synthetase (HLCS58). {yields} Full-length HLCS and HLCS58 enter the nucleus, but HLCS58 is the predominant variant. {yields} HLCS58 has biological activity as biotin protein ligase. -- Abstract: Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.

  13. Common genetic variants of the mitochondrial trafficking system and mitochondrial uncoupling proteins affect the development of two slowly developing demyelinating disorders, leukoaraiosis and multiple sclerosis.

    Science.gov (United States)

    Szolnoki, Z

    2010-01-01

    As the central energy source, the mitochondria are of great importance in the maintenance of the glia cells of the brain. It is presumed that mitochondrial energy production is affected not only by well-characterized genetic mutations of the mitochondria, which are associated with severe malfunctions and resultant acute glia and neuronal cell death, but also by a number of other unfavorable genetic variants. The genetic variants of the kinesin motor proteins and mitochondrial uncoupling proteins (UCPs) are believed to influence the mitochondrial energy production in different distress states of the glia cells. The kinesin motor proteins carry the mitochondria from the central parts to the peripheral parts of the glia cells, where myelin protein synthesis takes place. The UCPs are essential for regulation of the mitochondrial membrane potential under different physiological conditions, thereby finally attuning mitochondrial energy production in environmental states such as cold exposure, fasting or chronic mild hypoxia. While the capacity of the kinesin motor proteins can affect the number of mitochondria in the peripheral parts of the glia cells, the functional features of the UCPs can affect the degree of energy production of the mitochondria by influencing the mitochondrial membrane potential. The different genetic variants may display different activities, and some may result in a slowly developing energy shortage in the glia cells. In this context, this article discusses the roles of genetic variants of the kinesin motor proteins and UCPs in slowly developing diseases of the white matter of the brain as multiple sclerosis and leukoaraiosis.

  14. Melanopsin Variants as Intrinsic Optogenetic On and Off Switches for Transient versus Sustained Activation of G Protein Pathways.

    Science.gov (United States)

    Spoida, Katharina; Eickelbeck, Dennis; Karapinar, Raziye; Eckhardt, Tobias; Mark, Melanie D; Jancke, Dirk; Ehinger, Benedikt Valerian; König, Peter; Dalkara, Deniz; Herlitze, Stefan; Masseck, Olivia A

    2016-05-09

    G-protein-coupled receptors (GPCRs) represent the major protein family for cellular modulation in mammals. Therefore, various strategies have been developed to analyze the function of GPCRs involving pharmaco- and optogenetic approaches [1, 2]. However, a tool that combines precise control of the activation and deactivation of GPCR pathways and/or neuronal firing with limited phototoxicity is still missing. We compared the biophysical properties and optogenetic application of a human and a mouse melanopsin variant (hOpn4L and mOpn4L) on the control of Gi/o and Gq pathways in heterologous expression systems and mouse brain. We found that GPCR pathways can be switched on/off by blue/yellow light. The proteins differ in their kinetics and wavelength dependence to activate and deactivate G protein pathways. Whereas mOpn4L is maximally activated by very short light pulses, leading to sustained G protein activation, G protein responses of hOpn4L need longer light pulses to be activated and decline in amplitude. Based on the different biophysical properties, brief light activation of mOpn4L is sufficient to induce sustained neuronal firing in cerebellar Purkinje cells (PC), whereas brief light activation of hOpn4L induces AP firing, which declines in frequency over time. Most importantly, mOpn4L-induced sustained firing can be switched off by yellow light. Based on the biophysical properties, hOpn4L and mOpn4L represent the first GPCR optogenetic tools, which can be used to switch GPCR pathways/neuronal firing on an off with temporal precision and limited phototoxicity. We suggest to name these tools moMo and huMo for future optogenetic applications.

  15. E4orf6 variants with separate abilities to augment adenovirus replication and direct nuclear localization of the E1B 55-kilodalton protein.

    Science.gov (United States)

    Orlando, Joseph S; Ornelles, David A

    2002-02-01

    The E4orf6 protein of group C adenovirus is an oncoprotein that, in association with the E1B 55-kDa protein and by E1B-independent means, promotes virus replication. An arginine-faced amphipathic alpha-helix in the E4orf6 protein is required for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein and to enhance replication of an E4 deletion virus. In this study, E4orf6 protein variants containing arginine substitutions in the amphipathic alpha-helix were analyzed. Two of the six arginine residues within the alpha-helix, arginine-241 and arginine-243, were critical for directing nuclear localization of the E1B 55-kDa protein. The four remaining arginine residues appear to provide a net positive charge for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein. The molecular determinants of the arginine-faced amphipathic alpha-helix that were required for the functional interaction between the E4orf6 and E1B 55-kDa proteins seen in the transfected cell differed from those required to support a productive infection. Several E4orf6 protein variants with arginine-to-glutamic acid substitutions that failed to direct nuclear localization of the E1B 55-kDa protein restored replication of an E4 deletion virus. Additionally, a variant containing an arginine-to-alanine substitution at position 243 that directed nuclear localization of the E1B 55-kDa protein failed to enhance virus replication. These results indicate that the ability of the E4orf6 protein to relocalize the E1B 55-kDa protein to the nucleus can be separated from the ability of the E4orf6 protein to support a productive infection.

  16. Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant: A CANDIDATE MOLECULE FOR FUNGAL ALLERGY VACCINES.

    Science.gov (United States)

    Sircar, Gaurab; Jana, Kuladip; Dasgupta, Angira; Saha, Sudipto; Gupta Bhattacharya, Swati

    2016-08-19

    Efficacy of allergen-specific immunotherapy is often severely impaired by detrimental IgE-mediated side effects of native allergen during vaccination. Here, we present the molecular determinants for IgE recognition of Rhi o 1 and eventually converting the allergen into a hypoallergenic immunogen to restrain health hazards during desensitization. Rhi o 1 is a respiratory fungal allergen. Despite having cross-reactivity with cockroach allergen, we observed that non-cross-reactive epitope predominantly determined IgE binding to Rhi o 1. Denaturation and refolding behavior of the allergen confirmed that its IgE reactivity was not essentially conformation-dependent. A combinatorial approach consisting of computational prediction and a peptide-based immunoassay identified two peptides ((44)TGEYLTQKYFNSQRNN and (311)GAEKNWAGQYVVDCNK) of Rhi o 1 that frequently reacted with IgE antibodies of sensitized patients. Interestingly, these peptides did not represent purely linear IgE epitopes but were presented in a conformational manner by forming a spatially clustered surface-exposed epitope conferring optimal IgE-binding capacity to the folded allergen. Site-directed alanine substitution identified four residues of the IgE epitope that were crucial for antibody binding. A multiple mutant (T49A/Y52A/K314A/W316A) showing 100-fold lower IgE binding and reduced allergenic activity was generated. The TYKW mutant retained T-cell epitopes, as evident from its lymphoproliferative capacity but down-regulated pro-allergic IL-5 secretion. The TYKW mutant induced enhanced focusing of blocking IgG antibodies specifically toward the IgE epitope of the allergen. Anti-TYKW mutant polyclonal IgG antibodies competitively inhibited binding of IgE antibodies to Rhi o 1 up to 70% and suppressed allergen-mediated histamine release by 10-fold. In conclusion, this is a simple yet rational strategy based on epitope mapping data to develop a genetically modified hypoallergenic variant showing

  17. Repressed synthesis of ribosomal proteins generates protein-specific cell cycle and morphological phenotypes.

    Science.gov (United States)

    Thapa, Mamata; Bommakanti, Ananth; Shamsuzzaman, Md; Gregory, Brian; Samsel, Leigh; Zengel, Janice M; Lindahl, Lasse

    2013-12-01

    The biogenesis of ribosomes is coordinated with cell growth and proliferation. Distortion of the coordinated synthesis of ribosomal components affects not only ribosome formation, but also cell fate. However, the connection between ribosome biogenesis and cell fate is not well understood. To establish a model system for inquiries into these processes, we systematically analyzed cell cycle progression, cell morphology, and bud site selection after repression of 54 individual ribosomal protein (r-protein) genes in Saccharomyces cerevisiae. We found that repression of nine 60S r-protein genes results in arrest in the G2/M phase, whereas repression of nine other 60S and 22 40S r-protein genes causes arrest in the G1 phase. Furthermore, bud morphology changes after repression of some r-protein genes. For example, very elongated buds form after repression of seven 60S r-protein genes. These genes overlap with, but are not identical to, those causing the G2/M cell cycle phenotype. Finally, repression of most r-protein genes results in changed sites of bud formation. Strikingly, the r-proteins whose repression generates similar effects on cell cycle progression cluster in the ribosome physical structure, suggesting that different topological areas of the precursor and/or mature ribosome are mechanistically connected to separate aspects of the cell cycle.

  18. A region of the N-terminal domain of meningococcal factor H-binding protein that elicits bactericidal antibody across antigenic variant groups.

    Science.gov (United States)

    Beernink, Peter T; LoPasso, Carla; Angiolillo, Antonella; Felici, Franco; Granoff, Dan

    2009-05-01

    Meningococcal factor H-binding protein (fHbp) is a promising vaccine antigen. Previous studies described three fHbp antigenic variant groups and identified amino acid residues between 100 and 255 as important targets of variant-specific bactericidal antibodies. We investigated residues affecting expression of an epitope recognized by a murine IgG2a anti-fHbp mAb, designated JAR 4, which cross-reacted with fHbps in variant group 1 or 2 (95% of strains), and elicited human complement-mediated, cooperative bactericidal activity with other non-bactericidal anti-fHbp mAbs with epitopes involving residues between 121 and 216. From filamentous bacteriophage libraries containing random peptides that were recognized by JAR 4, we identified a consensus tripeptide, DHK that matched residues 25-27 in the N-terminal domain of fHbp. Since DHK was present in both JAR 4-reactive and non-reactive fHbps, the tripeptide was necessary but not sufficient for reactivity. Based on site-directed mutagenesis studies, the JAR 4 epitope could either be knocked out of a reactive variant 1 fHbp, or introduced into a non-reactive variant 3 protein. Collectively, the data indicated that the JAR 4 epitope was discontinuous and involved DHK residues beginning at position 25; YGN residues beginning at position 57; and a KDN tripeptide that was present in variant 3 proteins beginning at position 67 that negatively affected expression of the epitope. Thus, the region of fHbp encompassing residues 25-59 in the N-terminal domain is important for eliciting antibodies that can cooperate with other anti-fHbp antibodies for cross-reactive bactericidal activity against strains expressing fHbp from different antigenic variant groups.

  19. Extracellular matrix proteins expression profiling in chemoresistant variants of the A2780 ovarian cancer cell line.

    Science.gov (United States)

    Januchowski, Radosław; Zawierucha, Piotr; Ruciński, Marcin; Nowicki, Michał; Zabel, Maciej

    2014-01-01

    Ovarian cancer is the leading cause of death among gynaecological malignancies. Extracellular matrix (ECM) can affect drug resistance by preventing the penetration of the drug into cancer cells and increased resistance to apoptosis. This study demonstrates alterations in the expression levels of ECM components and related genes in cisplatin-, doxorubicin-, topotecan-, and paclitaxel-resistant variants of the A2780 ovarian cancer cell line. Affymetrix Gene Chip Human Genome Array Strips were used for hybridisations. The genes that had altered expression levels in drug-resistant sublines were selected and filtered by scatter plots. The genes that were up- or downregulated more than fivefold were selected and listed. Among the investigated genes, 28 genes were upregulated, 10 genes were downregulated, and two genes were down- or upregulated depending on the cell line. Between upregulated genes 12 were upregulated very significantly--over 20-fold. These genes included COL1A2, COL12A1, COL21A1, LOX, TGFBI, LAMB1, EFEMP1, GPC3, SDC2, MGP, MMP3, and TIMP3. Four genes were very significantly downregulated: COL11A1, LAMA2, GPC6, and LUM. The expression profiles of investigated genes provide a preliminary insight into the relationship between drug resistance and the expression of ECM components. Identifying correlations between investigated genes and drug resistance will require further analysis.

  20. Weaver Syndrome‐Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro

    Science.gov (United States)

    Yap, Damian B.; Lewis, M.E. Suzanne; Chijiwa, Chieko; Ramos‐Arroyo, Maria A.; Tkachenko, Natália; Milano, Valentina; Fradin, Mélanie; McKinnon, Margaret L.; Townsend, Katelin N.; Xu, Jieqing; Van Allen, M.I.; Ross, Colin J.D.; Dobyns, William B.; Weaver, David D.; Gibson, William T.

    2016-01-01

    ABSTRACT Weaver syndrome (WS) is a rare congenital disorder characterized by generalized overgrowth, macrocephaly, specific facial features, accelerated bone age, intellectual disability, and susceptibility to cancers. De novo mutations in the enhancer of zeste homolog 2 (EZH2) have been shown to cause WS. EZH2 is a histone methyltransferase that acts as the catalytic agent of the polycomb‐repressive complex 2 (PRC2) to maintain gene repression via methylation of lysine 27 on histone H3 (H3K27). Functional studies investigating histone methyltransferase activity of mutant EZH2 from various cancers have been reported, whereas WS‐associated mutations remain poorly characterized. To investigate the role of EZH2 in WS, we performed functional studies using artificially assembled PRC2 complexes containing mutagenized human EZH2 that reflected the codon changes predicted from patients with WS. We found that WS‐associated amino acid alterations reduce the histone methyltransferase function of EZH2 in this in vitro assay. Our results support the hypothesis that WS is caused by constitutional mutations in EZH2 that alter the histone methyltransferase function of PRC2. However, histone methyltransferase activities of different EZH2 variants do not appear to correlate directly with the phenotypic variability between WS patients and individuals with a common c.553G>C (p.Asp185His) polymorphism in EZH2. PMID:26694085

  1. Expression of a nonmyristylated variant of the catalytic subunit of protein kinase A during male germ-cell development.

    Science.gov (United States)

    Desseyn, J L; Burton, K A; McKnight, G S

    2000-06-06

    The catalytic subunits of protein kinase A are transcribed in all mouse tissues from two distinct genes that code for the Calpha and Cbeta isoforms. Alternative promoters exist for the Cbeta gene that are used in a tissue-specific fashion and give rise to variants that differ in their amino-terminal sequences. We have characterized an alternative promoter that is present in the first intron of the Calpha gene and is transcriptionally active in male germ cells. Transcription from this promoter is coincident with the appearance of pachytene spermatocytes and leads to a Calpha protein (Calpha2) that contains a distinctive 7 amino acid amino-terminus differing from the 14 amino acid amino-terminus of Calpha1. The Calpha2 protein does not contain the myristylation signal present on Calpha1 and migrates at a lower molecular weight on SDS/PAGE gels. By Western blotting, we estimate that most or all of the Calpha protein present in mature sperm is Calpha2. The amino-terminal sequence of Calpha2 is similar to that of ovine sperm C as previously reported [San Agustin, J. T., Leszyk, J. D., Nuwaysir, L. M. & Witman, G. B. (1998) J. Biol. Chem. 273, 24874-24883], and we show by cDNA cloning that human sperm also express a highly related Calpha2 homolog. The Calpha2 subunit forms holoenzymes with either RIIalpha or RIalpha, and both activate at the same concentration of cyclic nucleotide. Because protein kinase A is thought to play a pivotal role in sperm motility and capacitation, the distinctive biochemical properties of the unmyristylated Calpha2 may be essential for fertility in the male.

  2. A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Sara E. Howden

    2016-09-01

    Full Text Available While Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines, the CRISPR-Cas9 system can introduce undesirable “on-target” mutations within the second allele of successfully modified cells via non-homologous end joining (NHEJ. To address this, we fused the Streptococcus pyogenes Cas9 (SpCas9 nuclease to a peptide derived from the human Geminin protein (SpCas9-Gem to facilitate its degradation during the G1 phase of the cell cycle, when DNA repair by NHEJ predominates. We also use mRNA transfection to facilitate low and transient expression of modified and unmodified versions of Cas9. Although the frequency of homologous recombination was similar for SpCas9-Gem and SpCas9, we observed a marked reduction in the capacity for SpCas9-Gem to induce NHEJ-mediated indels at the target locus. Moreover, in contrast to native SpCas9, we demonstrate that transient SpCas9-Gem expression enables reliable generation of both knockin reporter cell lines and genetically repaired patient-specific induced pluripotent stem cell lines free of unwanted mutations at the targeted locus.

  3. Cloning and Characterization of Spliced Variants of the Porcine G Protein Coupled Receptor 120

    Directory of Open Access Journals (Sweden)

    Tongxing Song

    2015-01-01

    Full Text Available The polyunsaturated fatty acids (PUFAs receptor GPR120 exerts a significant impact on systemic nutrient homeostasis in human and rodents. However, the porcine GPR120 (pGPR120 has not been well characterized. In the current study, we found that pGPR120 had 3 spliced variants. Transcript 1 encoded 362-amino acids (aa wild type pGPR120-WT, which shared 88% homology with human short form GPR120. Transcript 1 was the mainly expressed transcript of pGPR120. It was expressed predominantly in ileum, jejunum, duodenum, spleen, and adipose. Transcript 3 (coding 320-aa isoform was detected in spleen, while the transcript 2 (coding 310-aa isoform was only slightly expressed in spleen. A selective agonist for human GPR120 (TUG-891 and PUFAs activated SRE-luc and NFAT-luc reporter in HEK293T cells transfected with construct for pGPR120-WT but not pGPR120-V2. However, 320-aa isoform was not a dominant negative isoform. The extracellular signal-regulated kinase 1/2 (ERK1/2 phosphorylation levels in cells transfected with construct for pGPR120-WT were well activated by PUFAs, especially n-3 PUFA. These results showed that although pGPR120 had 3 transcripts, transcript 1 which encoded pGPR120-WT was the mainly expressed transcript. TUG-891 and PUFAs, especially n-3 PUFA, well activated pGPR120-WT. The current study contributed to dissecting the molecular regulation mechanisms of n-3 PUFA in pigs.

  4. Low density lipoprotein receptor related protein 1 variant interacts with saturated fatty acids in Puerto Ricans

    Science.gov (United States)

    Low density lipoprotein related receptor protein 1 (LRP1) is a multi-functional endocytic receptor that is highly expressed in adipocytes and the hypothalamus. Animal models and in vitro studies support a role for LRP1 in adipocyte metabolism and leptin signaling, but genetic polymorphisms have not ...

  5. Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants.

    Science.gov (United States)

    Betti, Camilla; Lico, Chiara; Maffi, Dario; D'Angeli, Simone; Altamura, Maria Maddalena; Benvenuto, Eugenio; Faoro, Franco; Baschieri, Selene

    2012-02-01

    Potato virus X coat protein is necessary for both cell-to-cell and phloem transfer, but it has not been clarified definitively whether it is needed in both movement phases solely as a component of the assembled particles or also of differently structured ribonucleoprotein complexes. To clarify this issue, we studied the infection progression of a mutant carrying an N-terminal deletion of the coat protein, which was used to construct chimeric virus particles displaying peptides selectively affecting phloem transfer or cell-to-cell movement. Nicotiana benthamiana plants inoculated with expression vectors encoding the wild-type, mutant and chimeric viral genomes were examined by microscopy techniques. These experiments showed that coat protein-peptide fusions promoting cell-to-cell transfer only were not competent for virion assembly, whereas long-distance movement was possible only for coat proteins compatible with virus particle formation. Moreover, the ability of the assembled PVX to enter and persist into developing xylem elements was revealed here for the first time.

  6. C-reactive protein and genetic variants and cognitive decline in old age: The PROSPER Study

    Science.gov (United States)

    Plasma concentrations of C-reactive protein (CRP), a marker of chronic inflammation, have been associated with cognitive impairment in old age. However, it is unknown whether CRP is causally linked to cognitive decline. Within the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER) tri...

  7. Generation of mice lacking DUF1220 protein domains

    DEFF Research Database (Denmark)

    Keeney, J G; O'Bleness, M S; Anderson, N

    2015-01-01

    Sequences encoding DUF1220 protein domains show the most extreme human lineage-specific copy number increase of any coding region in the genome and have been linked to human brain evolution. In addition, DUF1220 copy number (dosage) has been implicated in influencing brain size within the human...... species, both in normal populations and in individuals associated with brain size pathologies (1q21-associated microcephaly and macrocephaly). More recently, increasing dosage of a subtype of DUF1220 has been linked with increasing severity of the primary symptoms of autism. Despite these intriguing...... associations, a function for these domains has not been described. As a first step in addressing this question, we have developed the first transgenic model of DUF1220 function by removing the single DUF1220 domain (the ancestral form) encoded in the mouse genome. In a hypothesis generating exercise...

  8. Generation of protein lattices by fusing proteins with matching rotational symmetry

    Science.gov (United States)

    Sinclair, John C.; Davies, Karen M.; Vénien-Bryan, Catherine; Noble, Martin E. M.

    2011-09-01

    The self-assembly of supramolecular structures that are ordered on the nanometre scale is a key objective in nanotechnology. DNA and peptide nanotechnologies have produced various two- and three-dimensional structures, but protein molecules have been underexploited in this area of research. Here we show that the genetic fusion of subunits from protein assemblies that have matching rotational symmetry generates species that can self-assemble into well-ordered, pre-determined one- and two-dimensional arrays that are stabilized by extensive intermolecular interactions. This new class of supramolecular structure provides a way to manufacture biomaterials with diverse structural and functional properties.

  9. The Role of the Carbohydrate Recognition Domain of Placental Protein 13 (PP13) in Pregnancy Evaluated with Recombinant PP13 and the DelT221 PP13 Variant

    OpenAIRE

    Marei Sammar; Shahar Nisamblatt; Ron Gonen; Berthold Huppertz; Sveinbjorn Gizurarson; George Osol; Hamutal Meiri

    2014-01-01

    Introduction Placental protein 13 (PP13), a placenta specific protein, is reduced in the first trimester of pregnancy in women who subsequently develop preeclampsia. A naturally occurring PP13 deletion of thymidine at position 221 (DelT221 or truncated variant) is associated with increased frequency of severe preeclampsia. In this study we compared the full length (wildtype) PP13 and the truncated variant. Methods Full length PP13 or its DelT221 variant were cloned, expressed and purified fro...

  10. Rare, Low-Frequency, and Common Variants in the Protein-Coding Sequence of Biological Candidate Genes from GWASs Contribute to Risk of Rheumatoid Arthritis

    NARCIS (Netherlands)

    Diogo, Dorothee; Kurreeman, Fina; Stahl, Eli A.; Liao, Katherine P.; Gupta, Namrata; Greenberg, Jeffrey D.; Rivas, Manuel A.; Hickey, Brendan; Flannick, Jason; Thomson, Brian; Guiducci, Candace; Ripke, Stephan; Adzhubey, Ivan; Barton, Anne; Kremer, Joel M.; Alfredsson, Lars; Sunyaev, Shamil; Martin, Javier; Zhernakova, Alexandra; Bowes, John; Eyre, Steve; Siminovitch, Katherine A.; Gregersen, Peter K.; Worthington, Jane; Klareskog, Lars; Padyukov, Leonid; Raychaudhuri, Soumya; Plenge, Robert M.

    2013-01-01

    The extent to which variants in the protein-coding sequence of genes contribute to risk of rheumatoid arthritis (RA) is unknown. In this study, we addressed this issue by deep exon sequencing and large-scale genotyping of 25 biological candidate genes located within RA risk loci discovered by genome

  11. Distinct composition of bovine milk from Jersey and Holstein-Friesian cows with good, poor or non-coagulation properties as reflected in protein genetic variants and isoforms

    DEFF Research Database (Denmark)

    Jensen, Hanne Bak; Poulsen, Nina Aagaard; Andersen, Kell Kleiner

    2012-01-01

    of minerals (Ca, P, Mg) were identified in poorly coagulating and noncoagulating milk in comparison with milk with good coagulation properties. Liquid chromatography/electrospray ionization-mass spectrometry revealed the presence of a great variety of genetic variants of the major milk proteins, namely, αS1...

  12. Interconverting conformations of variants of the human amyloidogenic protein beta2-microglobulin quantitatively characterized by dynamic capillary electrophoresis and computer simulation

    DEFF Research Database (Denmark)

    Heegaard, Niels H H; Jørgensen, Thomas J D; Cheng, Lei

    2006-01-01

    Capillary electrophoretic separation profiles of cleaved variants of beta2-microglobulin (beta2m) reflect the conformational equilibria existing in solutions of these proteins. The characterization of these equilibria is of interest since beta2m is responsible for amyloid formation in dialysis-re...

  13. Human T-cell recognition of synthetic peptides representing conserved and variant sequences from the merozoite surface protein 2 of Plasmodium falciparum

    DEFF Research Database (Denmark)

    Theander, T G; Hviid, L; Dodoo, D

    1997-01-01

    Merozoite surface protein 2 (MSP2) is a malaria vaccine candidate currently undergoing clinical trials. We analyzed the peripheral blood mononuclear cell (PBMC) response to synthetic peptides corresponding to conserved and variant regions of the FCQ-27 allelic form of MSP2 in Ghanaian individuals...

  14. Relationships between milk protein composition, milk protein variants, and cow fertility traits in Dutch Holstein-Friesian cattle

    NARCIS (Netherlands)

    Demeter, R.M.; Markiewicz, K.; Arendonk, van J.A.M.; Bovenhuis, H.

    2010-01-01

    Selective breeding can change milk protein composition to improve the manufacturing properties of milk. However, the effects of such breeding strategies on other economically important traits should be investigated before implementation. The objectives of this study were to examine the association b

  15. Rare, Low-Frequency, and Common Variants in the Protein-Coding Sequence of Biological Candidate Genes from GWASs Contribute to Risk of Rheumatoid Arthritis

    Science.gov (United States)

    Diogo, Dorothée; Kurreeman, Fina; Stahl, Eli A.; Liao, Katherine P.; Gupta, Namrata; Greenberg, Jeffrey D.; Rivas, Manuel A.; Hickey, Brendan; Flannick, Jason; Thomson, Brian; Guiducci, Candace; Ripke, Stephan; Adzhubey, Ivan; Barton, Anne; Kremer, Joel M.; Alfredsson, Lars; Sunyaev, Shamil; Martin, Javier; Zhernakova, Alexandra; Bowes, John; Eyre, Steve; Siminovitch, Katherine A.; Gregersen, Peter K.; Worthington, Jane; Klareskog, Lars; Padyukov, Leonid; Raychaudhuri, Soumya; Plenge, Robert M.

    2013-01-01

    The extent to which variants in the protein-coding sequence of genes contribute to risk of rheumatoid arthritis (RA) is unknown. In this study, we addressed this issue by deep exon sequencing and large-scale genotyping of 25 biological candidate genes located within RA risk loci discovered by genome-wide association studies (GWASs). First, we assessed the contribution of rare coding variants in the 25 genes to the risk of RA in a pooled sequencing study of 500 RA cases and 650 controls of European ancestry. We observed an accumulation of rare nonsynonymous variants exclusive to RA cases in IL2RA and IL2RB (burden test: p = 0.007 and p = 0.018, respectively). Next, we assessed the aggregate contribution of low-frequency and common coding variants to the risk of RA by dense genotyping of the 25 gene loci in 10,609 RA cases and 35,605 controls. We observed a strong enrichment of coding variants with a nominal signal of association with RA (p A [p.His266Gln]), and a noncoding variant, rs624988, reside on distinct haplotypes and independently contribute to the risk of RA (p = 4.6 × 10−6). Overall, our results indicate that variants (distributed across the allele-frequency spectrum) within the protein-coding portion of a subset of biological candidate genes identified by GWASs contribute to the risk of RA. Further, we have demonstrated that very large sample sizes will be required for comprehensively identifying the independent alleles contributing to the missing heritability of RA. PMID:23261300

  16. Differential interactions of cerebellin precursor protein (Cbln) subtypes and neurexin variants for synapse formation of cortical neurons.

    Science.gov (United States)

    Joo, Jae-Yeol; Lee, Sung-Jin; Uemura, Takeshi; Yoshida, Tomoyuki; Yasumura, Misato; Watanabe, Masahiko; Mishina, Masayoshi

    2011-03-25

    Trans-synaptic interaction of postsynaptic glutamate receptor δ2 and presynaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates synapse formation in the cerebellum [T. Uemura, S.J. Lee, M. Yasumura, T. Takeuchi, T. Yoshida, M. Ra, R. Taguchi, K. Sakimura, M. Mishina, Cell 141 (2010) 1068-1079]. This finding raises a question whether other Cbln family members interact with NRXNs to regulate synapse formation in the forebrain. Here, we showed that Cbln1 and Cbln2 induced presynaptic differentiation of cultured cortical neurons, while Cbln4 exhibited little activity. When compared with neuroligin 1, Cbln1 and Cbln2 induced preferentially inhibitory presynaptic differentiation rather than excitatory one in cortical cultures. The synaptogenic activities of Cbln1 and Cbln2 were suppressed by the addition of the extracellular domain of NRXN1β to the cortical neuron cultures. Consistently, Cbln1 and Cbln2 showed robust binding activities to NRXN1α and three β-NRXNs, while only weak interactions were observed between Cbln4 and NRXNs. The interactions of Cbln1, Cbln2 and Cbln4 were selective for NRXN variants containing splice segment (S) 4. Affinities for NRXNs estimated by surface plasmon resonance analysis were variable among Cbln subtypes. Cbln1 showed higher affinities to NRXNs than Cbln2, while the binding ability of Cbln4 was much lower than those of Cbln1 and Cbln2. The affinities of Cbln1 and Cbln2 were comparable between NRXN1α and NRXN1β, but those for NRXN2β and NRXN3β were lower. These results suggest that Cbln subtypes exert synaptogenic activities in cortical neurons by differentially interacting with NRXN variants containing S4.

  17. Effect of enhanced Renilla luciferase and fluorescent protein variants on the Foerster distance of Bioluminescence resonance energy transfer (BRET)

    Energy Technology Data Exchange (ETDEWEB)

    Dacres, Helen, E-mail: helen.dacres@csiro.au [CSIRO Food Futures Flagship and Ecosystem Sciences, Canberra (Australia); Michie, Michelle; Wang, Jian [CSIRO Food Futures Flagship and Ecosystem Sciences, Canberra (Australia); Pfleger, Kevin D.G. [Laboratory for Molecular Endocrinology-GPCRs, Western Australian Institute for Medical Research (WAIMR) and Centre for Medical Research, The University of Western Australia, Perth (Australia); Trowell, Stephen C. [CSIRO Food Futures Flagship and Ecosystem Sciences, Canberra (Australia)

    2012-08-31

    Highlights: Black-Right-Pointing-Pointer First experimental determination of Foerster distance (R{sub 0}) for enhanced BRET systems. Black-Right-Pointing-Pointer Effect of brighter BRET components RLuc2, RLuc8 and Venus was assessed. Black-Right-Pointing-Pointer Using brighter BRET components substantially increased (25%) R{sub 0} of the BRET{sup 1} system. Black-Right-Pointing-Pointer Using brighter BRET components marginally increased (2-9%) R{sub 0} of the BRET{sup 2} system. Black-Right-Pointing-Pointer Brighter BRET components improve the different weaknesses of BRET{sup 1} and BRET{sup 2} systems. -- Abstract: Bioluminescence resonance energy transfer (BRET) is an important tool for monitoring macromolecular interactions and is useful as a transduction technique for biosensor development. Foerster distance (R{sub 0}), the intermolecular separation characterized by 50% of the maximum possible energy transfer, is a critical BRET parameter. R{sub 0} provides a means of linking measured changes in BRET ratio to a physical dimension scale and allows estimation of the range of distances that can be measured by any donor-acceptor pair. The sensitivity of BRET assays has recently been improved by introduction of new BRET components, RLuc2, RLuc8 and Venus with improved quantum yields, stability and brightness. We determined R{sub 0} for BRET{sup 1} systems incorporating novel RLuc variants RLuc2 or RLuc8, in combination with Venus, as 5.68 or 5.55 nm respectively. These values were approximately 25% higher than the R{sub 0} of the original BRET{sup 1} system. R{sub 0} for BRET{sup 2} systems combining green fluorescent proteins (GFP{sup 2}) with RLuc2 or RLuc8 variants was 7.67 or 8.15 nm, i.e. only 2-9% greater than the original BRET{sup 2} system despite being {approx}30-fold brighter.

  18. Protein C Thr315Ala variant results in gain of function but manifests as type II deficiency in diagnostic assays.

    Science.gov (United States)

    Ding, Qiulan; Yang, Likui; Dinarvand, Peyman; Wang, Xuefeng; Rezaie, Alireza R

    2015-04-09

    Protein C (PC) is a vitamin K-dependent plasma glycoprotein, which upon activation by thrombin in complex with thrombomodulin (TM), regulates the coagulation cascade through a feedback loop inhibition mechanism. PC deficiency is associated with an increased risk of venous thromboembolism (VTE). A recent cohort study aimed at establishing a normal PC range identified a healthy PC-deficient subject whose PC antigen level of 65% and activity levels of 50% (chromogenic assay) and 36% (clotting assay) were markedly low. The proband has a negative family history of VTE. Genetic analysis revealed the proband has a heterozygous missense mutation in which Thr-315 of the PC heavy chain has been substituted with Ala. We expressed this mutant in HEK-293 cells and purified it to homogeneity. A similar decrease in both anticoagulant and anti-inflammatory activities of the activated protein C mutant was observed in plasma- and cell-based assays. Interestingly, we discovered if functional assays were coupled to PC activation by the thrombin-TM complex, the variant exhibits improved activities in all assays. Sequence analysis revealed Thr-315 is a consensus N-linked glycosylation site for Asn-313 and that its elimination significantly (∼four- to fivefold) improves the maximum velocity of PC activation by the thrombin-TM complex, explaining the basis for the proband's negative VTE pedigree. © 2015 by The American Society of Hematology.

  19. Structural Rigidity and Protein Thermostability in Variants of Lipase A from Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Prakash Chandra Rathi

    Full Text Available Understanding the origin of thermostability is of fundamental importance in protein biochemistry. Opposing views on increased or decreased structural rigidity of the folded state have been put forward in this context. They have been related to differences in the temporal resolution of experiments and computations that probe atomic mobility. Here, we find a significant (p = 0.004 and fair (R2 = 0.46 correlation between the structural rigidity of a well-characterized set of 16 mutants of lipase A from Bacillus subtilis (BsLipA and their thermodynamic thermostability. We apply the rigidity theory-based Constraint Network Analysis (CNA approach, analyzing directly and in a time-independent manner the statics of the BsLipA mutants. We carefully validate the CNA results on macroscopic and microscopic experimental observables and probe for their sensitivity with respect to input structures. Furthermore, we introduce a robust, local stability measure for predicting thermodynamic thermostability. Our results complement work that showed for pairs of homologous proteins that raising the structural stability is the most common way to obtain a higher thermostability. Furthermore, they demonstrate that related series of mutants with only a small number of mutations can be successfully analyzed by CNA, which suggests that CNA can be applied prospectively in rational protein design aimed at higher thermodynamic thermostability.

  20. A Solution NMR Investigation into the Murine Amelogenin Splice-Variant LRAP (Leucine-Rich Amelogenin Protein).

    Energy Technology Data Exchange (ETDEWEB)

    Buchko, Garry W.; Tarasevich, Barbara J.; Roberts, Jacky; Snead, Malcolm L.; Shaw, Wendy J.

    2010-09-01

    Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up >90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the 1H-15N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like the full-length protein, is intrinsically disordered under these solution conditions. The major difference between the 1H-15N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12* and Y12 at the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggest that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region and is likely due to a slow exchange process. As observed with rp(H)M180, residue specific changes in molecular dynamics, manifested by the reduction in intensity and disappearance of 1H-15N HSQC cross peaks, were observed with the addition of NaCl to rp(H)LRAP. These perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different pattern of 1H-15N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences account for the cell signaling properties attributable to LRAP but not the full-length protein.

  1. Effect of Protein Charge on the Generation of Aggregation-Prone Conformers

    NARCIS (Netherlands)

    Broersen, K.; Weijers, M.; Groot, de J.; Hamer, R.J.; Jongh, de H.H.J.

    2007-01-01

    This study describes how charge modification affects aggregation of ovalbumin, thereby distinguishing the role of conformational and electrostatic stability in the process. Ovalbumin variants were engineered using chemical methylation or succinylation to obtain a range of protein net charge from -1

  2. Effect of protein charge on the generation of aggregation-prone conformers

    NARCIS (Netherlands)

    Broersen, K.; Weijers, M.; Groot, J.de; Hamer, R.J.; Jongh, H.H.J.de

    2007-01-01

    This study describes how charge modification affects aggregation of ovalbumin, thereby distinguishing the role of conformational and electrostatic stability in the process. Ovalbumin variants were engineered using chemical methylation or succinylation to obtain a range of protein net charge from -1

  3. Evidence for allosteric variants of wild-type p53, a tumour suppressor protein.

    OpenAIRE

    1990-01-01

    A tumour suppressor function for p53 is indicated in human lung cancer and in carcinoma of the colorectum. Loss of suppressor function, by mutation of the p53 gene, is associated with activation of p53 as an oncogene. The suppressor (wild type) and oncogenic (mutant) forms of the murine p53 protein are distinguishable at the molecular level by reactivity with anti-p53 monoclonal antibodies. For example, activated mutant p53 fails to react with PAb246 (p53-246 degrees). We now demonstrate that...

  4. A microRNA derived from an apparent canonical biogenesis pathway regulates variant surface protein gene expression in Giardia lamblia

    Science.gov (United States)

    Saraiya, Ashesh A.; Li, Wei; Wang, Ching C.

    2011-01-01

    We have previously shown that a snoRNA-derived microRNA, miR2, in Giardia lamblia potentially regulates the expression of 22 variant surface protein (VSP) genes. Here, we identified another miRNA, miR4, also capable of regulating the expression of several VSPs but derived from an unannotated open reading frame (ORF) rather than a snoRNA, suggesting a canonical miRNA biogenesis pathway in Giardia. miR4 represses expression of a reporter containing two miR4 antisense sequences at the 3′ UTR without causing a corresponding decrease in the mRNA level. This repression requires the presence of the Giardia Argonaute protein (GlAgo) and is reversed by 2′ O–methylated antisense oligo to miR4, suggesting an RNA-induced silencing complex (RISC)–mediated mechanism. Furthermore, in vivo and in vitro evidence suggested that the Giardia Dicer protein (GlDcr) is required for miR4 biogenesis. Coimmunoprecipitation of miR4 with GlAgo further verified miR4 as a miRNA. A total of 361 potential target sites for miR4 were bioinformatically identified in Giardia, out of which 69 (32.7%) were associated with VSP genes. miR4 reduces the expression of a reporter containing two copies of the target site from VSP (GL50803_36493) at the 3′ UTR. Sixteen of the 69 VSP genes were further found to contain partially overlapping miR2 and miR4 targeting sites. Expression of a reporter carrying the two overlapping sites was inhibited by either miR2 or miR4, but the inhibition was neither synergistic nor additive, suggesting a complex mechanism of miRNA regulation of VSP expression and the presence of a rich miRNAome in Giardia. PMID:22033329

  5. Analysis of Low Frequency Protein Truncating Stop-Codon Variants and Fasting Concentration of Growth Hormone.

    Directory of Open Access Journals (Sweden)

    Erik Hallengren

    Full Text Available The genetic background of Growth Hormone (GH secretion is not well understood. Mutations giving rise to a stop codon have a high likelihood of affecting protein function.To analyze likely functional stop codon mutations that are associated with fasting plasma concentration of Growth Hormone.We analyzed stop codon mutations in 5451 individuals in the Malmö Diet and Cancer study by genotyping the Illumina Exome Chip. To enrich for stop codon mutations with likely functional effects on protein function, we focused on those disrupting >80% of the predicted amino acid sequence, which were carried by ≥ 10 individuals. Such mutations were related to GH concentration, measured with a high sensitivity assay (hs-GH and, if nominally significant, to GH related phenotypes, using linear regression analysis.Two stop codon mutations were associated with the fasting concentration of hs-GH. rs121909305 (NP_005370.1:p.R93* [Minor Allele Frequency (MAF = 0.8%] in the Myosin 1A gene (MYO1A was associated with a 0.36 (95%CI, 0.04 to 0.54; p=0.02 increment of the standardized value of the natural logarithm of hs-GH per 1 minor allele and rs35699176 (NP_067040.1:p.Q100* in the Zink Finger protein 77 gene (ZNF77 (MAF = 4.8% was associated with a 0.12 (95%CI, 0.02 to 0.22; p = 0.02 increase of hs-GH. The mutated high hs-GH associated allele of MYO1A was related to lower BMI (β-coefficient, -0.22; p = 0.05, waist (β-coefficient, -0.22; p = 0.04, body fat percentage (β-coefficient, -0.23; p = 0.03 and with higher HDL (β-coefficient, 0.23; p = 0.04. The ZNF77 stop codon was associated with height (β-coefficient, 0.11; p = 0.02 but not with cardiometabolic risk factors.We here suggest that a stop codon of MYO1A, disrupting 91% of the predicted amino acid sequence, is associated with higher hs-GH and GH-related traits suggesting that MYO1A is involved in GH metabolism and possibly body fat distribution. However, our results are preliminary and need replication in

  6. Proteome analysis of protein partners to nucleosomes containing canonical H2A or the variant histones H2A.Z or H2A.X.

    Science.gov (United States)

    Fujimoto, Satoru; Seebart, Corrine; Guastafierro, Tiziana; Prenni, Jessica; Caiafa, Paola; Zlatanova, Jordanka

    2012-01-01

    Although the existence of histone variants has been known for quite some time, only recently are we grasping the breadth and diversity of the cellular processes in which they are involved. Of particular interest are the two variants of histone H2A, H2A.Z and H2A.X because of their roles in regulation of gene expression and in DNA double-strand break repair, respectively. We hypothesize that nucleosomes containing these variants may perform their distinct functions by interacting with different sets of proteins. Here, we present our proteome analysis aimed at identifying protein partners that interact with nucleosomes containing H2A.Z, H2A.X or their canonical H2A counterpart. Our development of a nucleosome-pull down assay and analysis of the recovered nucleosome-interacting proteins by mass spectrometry allowed us to directly compare nuclear partners of these variant-containing nucleosomes to those containing canonical H2A. To our knowledge, our data represent the first systematic analysis of the H2A.Z and H2A.X interactome in the context of nucleosome structure.

  7. Systematic generation of in vivo G protein-coupled receptor mutants in the rat.

    Science.gov (United States)

    van Boxtel, R; Vroling, B; Toonen, P; Nijman, I J; van Roekel, H; Verheul, M; Baakman, C; Guryev, V; Vriend, G; Cuppen, E

    2011-10-01

    G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies.

  8. Functional promoter variant in zinc finger protein 202 predicts severe atherosclerosis and ischemic heart disease

    DEFF Research Database (Denmark)

    Frikke-Schmidt, R.; Nordestgaard, Børge; Grande, Peer

    2008-01-01

    Objectives This study was designed to test the hypotheses that single nucleotide polymorphisms ( SNPs), in zinc finger protein 202 ( ZNF202), predict severe atherosclerosis and ischemic heart disease ( IHD). Background ZNF202 is a transcriptional repressor controlling promoter elements in genes......,998 controls. Finally, we determined whether g. -660A>G altered transcriptional activity of the ZNF202 promoter in vitro. Results Cross-sectionally, ZNF202 g. -660 GG versus AA homozygosity predicted an odds ratio for severe atherosclerosis of 2.01 ( 95% confidence interval [CI]: 1.34 to 3.01). Prospectively...... were highly correlated with g. -660A>G, also predicted risk of severe atherosclerosis and IHD. Finally, ZNF202 g. -660G versus g. -660A was associated with a 60% reduction in transcriptional activity in vitro, whereas none of the 2 correlated SNPs were predicted to be functional. Conclusions...

  9. Functional analysis of promoter variants in the microsomal triglyceride transfer protein (MTTP) gene.

    Science.gov (United States)

    Rubin, Diana; Schneider-Muntau, Alexandra; Klapper, Maja; Nitz, Inke; Helwig, Ulf; Fölsch, Ulrich R; Schrezenmeir, Jürgen; Döring, Frank

    2008-01-01

    The microsomal triglyceride transfer protein (MTTP) is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins from the intestine and liver. According to this function, polymorphic sites in the MTTP gene showed associations to low-density lipoprotein (LDL) cholesterol and related traits of the metabolic syndrome. Here we studied the functional impact of common MTTP promoter polymorphisms rs1800804:T>C (-164T>C), rs1800803:A>T (-400A>T), and rs1800591:G>T (-493G>T) using gene-reporter assays in intestinal Caco-2 and liver Huh-7 cells. Significant results were obtained in Huh-7 cells. The common MTTP promoter haplotype -164T/-400A/-493G showed about two-fold lower activity than the rare haplotype -164C/-400T/-493T. MTTP promoter mutant constructs -164T/-400A/-493T and -164T/-400T/-493T exhibited similar activity than the common haplotype. Activities of mutants -164C/-400A/-493G and -164C/-400A/-493T resembled the rare MTTP promoter haplotype. Electrophoretic mobility shift assays (EMSAs) revealed higher binding capacity of the transcriptional factor Sterol regulatory element binding protein1a (SREBP1a) to the -164T probe in comparison to the -164C probe. In conclusion, our study indicates that the polymorphism -164T>C mediates different activities of common MTTP promoter haplotypes via SREBP1a. This suggested that the already described SREBP-dependent modulation of MTTP expression by diet is more effective in -164T than in -164C carriers.

  10. Comparative analysis of the folding dynamics and kinetics of an engineered knotted protein and its variants derived from HP0242 of Helicobacter pylori

    Science.gov (United States)

    Wang, Liang-Wei; Liu, Yu-Nan; Lyu, Ping-Chiang; Jackson, Sophie E.; Hsu, Shang-Te Danny

    2015-09-01

    Understanding the mechanism by which a polypeptide chain thread itself spontaneously to attain a knotted conformation has been a major challenge in the field of protein folding. HP0242 is a homodimeric protein from Helicobacter pylori with intertwined helices to form a unique pseudo-knotted folding topology. A tandem HP0242 repeat has been constructed to become the first engineered trefoil-knotted protein. Its small size renders it a model system for computational analyses to examine its folding and knotting pathways. Here we report a multi-parametric study on the folding stability and kinetics of a library of HP0242 variants, including the trefoil-knotted tandem HP0242 repeat, using far-UV circular dichroism and fluorescence spectroscopy. Equilibrium chemical denaturation of HP0242 variants shows the presence of highly populated dimeric and structurally heterogeneous folding intermediates. Such equilibrium folding intermediates retain significant amount of helical structures except those at the N- and C-terminal regions in the native structure. Stopped-flow fluorescence measurements of HP0242 variants show that spontaneous refolding into knotted structures can be achieved within seconds, which is several orders of magnitude faster than previously observed for other knotted proteins. Nevertheless, the complex chevron plots indicate that HP0242 variants are prone to misfold into kinetic traps, leading to severely rolled-over refolding arms. The experimental observations are in general agreement with the previously reported molecular dynamics simulations. Based on our results, kinetic folding pathways are proposed to qualitatively describe the complex folding processes of HP0242 variants.

  11. Computational studies on receptor-ligand interactions between novel buffalo (Bubalus bubalis) nucleotide-binding oligomerization domain-containing protein 2 (NOD2) variants and muramyl dipeptide (MDP).

    Science.gov (United States)

    Brahma, Biswajit; Patra, Mahesh Chandra; Mishra, Purusottam; De, Bidhan Chandra; Kumar, Sushil; Maharana, Jitendra; Vats, Ashutosh; Ahlawat, Sonika; Datta, Tirtha Kumar; De, Sachinandan

    2016-04-01

    Nucleotide binding and oligomerization domain 2 (NOD2), a member of intracellular NOD-like receptors (NLRs) family, recognizes the bacterial peptidoglycan, muramyl dipeptide (MDP) and initiates host immune response. The precise ligand recognition mechanism of NOD2 has remained elusive, although studies have suggested leucine rich repeat (LRR) region of NOD2 as the possible binding site of MDP. In this study, we identified multiple transcripts of NOD2 gene in buffalo (buNOD2) and at least five LRR variants (buNOD2-LRRW (wild type), buNOD2-LRRV1-V4) were found to be expressed in buffalo peripheral blood mononuclear cells. The newly identified buNOD2 transcripts were shorter in lengths as a result of exon-skipping and frame-shift mutations. Among the variants, buNOD2-LRRW, V1, and V3 were expressed more frequently in the animals studied. A comparative receptor-ligand interaction study through modeling of variants, docking, and molecular dynamics simulation revealed that the binding affinity of buNOD2-LRRW towards MDP was greater than that of the shorter variants. The absence of a LRR segment in the buNOD2 variants had probably affected their affinity toward MDP. Notwithstanding a high homology among the variants, the amino acid residues that interact with MDP were located on different LRR motifs. The binding free energy calculation revealed that the amino acids Arg850(LRR4) and Glu932(LRR7) of buNOD2-LRRW, Lys810(LRR3) of buNOD2-LRRV1, and Lys830(LRR3) of buNOD2-LRRV3 largely contributed towards MDP recognition. The knowledge of MDP recognition and binding modes on buNOD2 variants could be useful to understand the regulation of NOD-mediated immune response as well as to develop next generation anti-inflammatory compounds.

  12. Common FABP4 Genetic Variants and Plasma Levels of Fatty Acid Binding Protein 4 in Older Adults

    Science.gov (United States)

    Mukamal, Kenneth J.; Wilk, Jemma B.; Biggs, Mary L.; Jensen, Majken K.; Ix, Joachim H.; Kizer, Jorge R.; Tracy, Russell P.; Zieman, Susan J.; Mozaffarian, Dariush; Psaty, Bruce M.; Siscovick, David S.; Djoussé, Luc

    2013-01-01

    We examined common variants in the fatty acid binding protein 4 gene (FABP4) and plasma levels of FABP4 in adults aged 65 and older from the Cardiovascular Health Study. We genotyped rs16909187, rs1054135, rs16909192, rs10808846, rs7018409, rs2290201, and rs6992708 and measured circulating FABP4 levels among 3190 European Americans and 660 African Americans. Among European Americans, the minor alleles of six single nucleotide polymorphisms (SNP) were associated with lower FABP4 levels (all p ≤ 0.01). Among African Americans, the SNP with the lowest minor allele frequency was associated with lower FABP4 levels (p = 0.015). The C-A haplotype of rs16909192 and rs2290201 was associated with lower FABP4 levels in both European Americans (frequency = 16 %; p = 0.001) and African Americans (frequency = 8 %; p = 0.04). The haplotype combined a SNP in the first intron with one in the 3′untranslated region. However, the alleles associated with lower FABP4 levels were associated with higher fasting glucose in meta-analyses from the MAGIC consortium. These results demonstrate associations of common SNP and haplotypes in the FABP4 gene with lower plasma FABP4 but higher fasting glucose levels. PMID:24043587

  13. Genetic variants of uncoupling proteins-2 and -3 in relation to maximal oxygen uptake in different sports.

    Science.gov (United States)

    Holdys, Joanna; Gronek, Piotr; Kryściak, Jakub; Stanisławski, Daniel

    2013-01-01

    Uncoupling proteins 2 and 3 (UCP2 and UCP3) as mitochondrial electron transporters are involved in regulation of ATP production and energy dissipation as heat. Energy efficiency plays an important role in physical performance, especially in aerobic fitness. The aim of this study was to examine the association between maximal oxygen uptake and genetic variants of the UCP2 and UCP3 genes. The studies were carried out in a group of 154 men and 85 women, professional athletes representing various sports and fitness levels and students of the University of Physical Education in Poznań. Physiological and molecular procedures were used, i.e. direct measurement of maximum oxygen uptake (VO₂max) and analysis of an insertion/deletion (I/D) polymorphism in the 3'untranslated region of exon 8 of the UCP2 gene and a C>T substitution in exon 5 (Y210Y) of the UCP3 gene. No statistically significant associations were found, only certain trends. Insertion allele (I) of the I/D UCP2 and the T allele of the UCP3 gene were favourable in obtaining higher VO₂max level and might be considered as endurance-related alleles.

  14. Variants of tumor necrosis factor-induced protein 3 gene are associated with left ventricular hypertrophy in hypertensive patients

    Institute of Scientific and Technical Information of China (English)

    XUE Hao; WANG Shu-xia; WANG Xiao-jian; XIN Ying; WANG Hu; SONG Xiao-dong; SUN Kai; WANG Yi-bo; HUI Ru-tai

    2011-01-01

    Background Tumor necrosis factor-induced protein 3 (TNFAIP3) gene has been shown important in cardiac remodeling. The aim of the present study was to investigate whether the variants of TNFAIP3 gene are associated with left ventricular hypertrophy (LVH) in hypertensive patients.Methods Four representatives of all the other single nucleotide polymorphisms (SNPs) in TNFAIP3 gene were tested for association with hypertrophy in two independent hypertensive populations (n=2120 and n=324).Results We found that only the tag SNP (rs5029939) was consistently lower in the hypertensives with cardiac hypertrophy than in those without cardiac hypertrophy in the two study populations, indicating a protective effect on LVH (odds ratio (OR) (95% confidence interval (CI))0.58 (0.358-0.863), P=0.035; OR (95% CI)=0.477 (0.225-0.815), P<0.05,respectively). Multiple regression analyses confirmed that the patients with G allele of rs5029939 had less thickness in inter-ventricular septum, left ventricular posterior wall, relative wall thickness and left ventricular mass index than did those with CC allele in the hypertensive patients in both study populations (all P<0.01).Conclusion These findings indicate that the SNP (rs5029939) in the TNFAIP3 gene may serve as a novel protective genetic marker for the development of LVH in patients with hypertension.

  15. One-step design of a stable variant of the malaria invasion protein RH5 for use as a vaccine immunogen

    Science.gov (United States)

    Campeotto, Ivan; Goldenzweig, Adi; Davey, Jack; Barfod, Lea; Marshall, Jennifer M.; Silk, Sarah E.; Wright, Katherine E.; Higgins, Matthew K.; Fleishman, Sarel J.

    2017-01-01

    Many promising vaccine candidates from pathogenic viruses, bacteria, and parasites are unstable and cannot be produced cheaply for clinical use. For instance, Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is essential for erythrocyte invasion, is highly conserved among field isolates, and elicits antibodies that neutralize in vitro and protect in an animal model, making it a leading malaria vaccine candidate. However, functional RH5 is only expressible in eukaryotic systems and exhibits moderate temperature tolerance, limiting its usefulness in hot and low-income countries where malaria prevails. Current approaches to immunogen stabilization involve iterative application of rational or semirational design, random mutagenesis, and biochemical characterization. Typically, each round of optimization yields minor improvement in stability, and multiple rounds are required. In contrast, we developed a one-step design strategy using phylogenetic analysis and Rosetta atomistic calculations to design PfRH5 variants with improved packing and surface polarity. To demonstrate the robustness of this approach, we tested three PfRH5 designs, all of which showed improved stability relative to wild type. The best, bearing 18 mutations relative to PfRH5, expressed in a folded form in bacteria at >1 mg of protein per L of culture, and had 10–15 °C higher thermal tolerance than wild type, while also retaining ligand binding and immunogenic properties indistinguishable from wild type, proving its value as an immunogen for a future generation of vaccines against the malaria blood stage. We envision that this efficient computational stability design methodology will also be used to enhance the biophysical properties of other recalcitrant vaccine candidates from emerging pathogens. PMID:28096331

  16. Expression of a second ecto-5'-nucleotidase variant besides the usual protein in symptomatic phase of experimental autoimmune encephalomyelitis.

    Science.gov (United States)

    Lavrnja, Irena; Laketa, Danijela; Savic, Danijela; Bozic, Iva; Bjelobaba, Ivana; Pekovic, Sanja; Nedeljkovic, Nadezda

    2015-04-01

    Ecto-5'-nucleotidase/cluster of differentiation 73 (CD73) (eN) is a 70-kDa glycoprotein expressed in several different mammalian tissues and cell types. It is the rate-limiting enzyme of the purine catabolic pathway, which catalyzes the hydrolysis of AMP to produce adenosine with known anti-inflammatory and immunosuppressive actions. There is strong evidence for lymphocyte and endothelial cell eN having a role in experimental autoimmune encephalomyelitis (EAE), but the role of eN in cell types within the central nervous system is less clear. We have previously shown that eN activity significantly increased in the lumbar spinal cord during EAE. The present study is aimed to explore molecular pattern of the eN upregulation over the course of the disease and cell type(s) accountable for the induction. EAE was induced in Dark Agouti (DA) rats by immunization with the spinal cord tissue homogenate and adjuvant. Animals were sacrificed 8, 15, and 28 days following immunization (D8, D15, and D28), i.e., at time points which corresponded to the presymptomatic, symptomatic, and postsymptomatic phases of the disease, respectively. Significant increase in eN activity and its upregulation at the gene and the protein levels were demonstrated at D15 and less prominently at D28 in comparison to control. Additionally, reactive astrocytes abundantly present in the lumbar spinal cord parenchyma were identified as principal cell type with significantly elevated eN expression. In all experimental groups, eN was expressed as a 71-kDa protein band of uniform abundance, whereas the overexpression of eN at D15 and D28 was associated with the expression of a second 75-kDa eN variant. The possible outcome of eN upregulation during EAE as a part of protective astrocyte repertoire contributing to the resolution of the disease is discussed.

  17. Identification of genetic risk variants for deep vein thrombosis by multiplexed next-generation sequencing of 186 hemostatic/pro-inflammatory genes

    Directory of Open Access Journals (Sweden)

    Lotta Luca A

    2012-02-01

    Full Text Available Abstract Background Next-generation DNA sequencing is opening new avenues for genetic association studies in common diseases that, like deep vein thrombosis (DVT, have a strong genetic predisposition still largely unexplained by currently identified risk variants. In order to develop sequencing and analytical pipelines for the application of next-generation sequencing to complex diseases, we conducted a pilot study sequencing the coding area of 186 hemostatic/proinflammatory genes in 10 Italian cases of idiopathic DVT and 12 healthy controls. Results A molecular-barcoding strategy was used to multiplex DNA target capture and sequencing, while retaining individual sequence information. Genomic libraries with barcode sequence-tags were pooled (in pools of 8 or 16 samples and enriched for target DNA sequences. Sequencing was performed on ABI SOLiD-4 platforms. We produced > 12 gigabases of raw sequence data to sequence at high coverage (average: 42X the 700-kilobase target area in 22 individuals. A total of 1876 high-quality genetic variants were identified (1778 single nucleotide substitutions and 98 insertions/deletions. Annotation on databases of genetic variation and human disease mutations revealed several novel, potentially deleterious mutations. We tested 576 common variants in a case-control association analysis, carrying the top-5 associations over to replication in up to 719 DVT cases and 719 controls. We also conducted an analysis of the burden of nonsynonymous variants in coagulation factor and anticoagulant genes. We found an excess of rare missense mutations in anticoagulant genes in DVT cases compared to controls and an association for a missense polymorphism of FGA (rs6050; p = 1.9 × 10-5, OR 1.45; 95% CI, 1.22-1.72; after replication in > 1400 individuals. Conclusions We implemented a barcode-based strategy to efficiently multiplex sequencing of hundreds of candidate genes in several individuals. In the relatively small dataset of

  18. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Albariño, César G., E-mail: calbarino@cdc.gov; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-10-15

    Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies.

  19. Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein

    Science.gov (United States)

    Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Enríquez-Flores, Sergio; De la Mora-De la Mora, Ignacio; González-Valdez, Abigail; García-Torres, Itzhel; Martínez-Rosas, Víctor; Sierra-Palacios, Edgar; Lazcano-Pérez, Fernando; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency), and the G6PD Santa Maria and A+ (less severe deficiency) (Class I, II and III, respectively) affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients. PMID:26633385

  20. Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein

    Directory of Open Access Journals (Sweden)

    Saúl Gómez-Manzo

    2015-12-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency, and the G6PD Santa Maria and A+ (less severe deficiency (Class I, II and III, respectively affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients.

  1. ICO amplicon NGS data analysis: a Web tool for variant detection in common high-risk hereditary cancer genes analyzed by amplicon GS Junior next-generation sequencing.

    Science.gov (United States)

    Lopez-Doriga, Adriana; Feliubadaló, Lídia; Menéndez, Mireia; Lopez-Doriga, Sergio; Morón-Duran, Francisco D; del Valle, Jesús; Tornero, Eva; Montes, Eva; Cuesta, Raquel; Campos, Olga; Gómez, Carolina; Pineda, Marta; González, Sara; Moreno, Victor; Capellá, Gabriel; Lázaro, Conxi

    2014-03-01

    Next-generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user-friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories.

  2. Monocyte chemoattractant protein-1 in spinal tuberculosis: -362G/C genetic variant and protein levels in Chinese patients.

    Science.gov (United States)

    Guo, Chaofeng; Zhang, Hongqi; Gao, Qile; He, Dan; Tang, Mingxing; Liu, Shaohua; Deng, Ang; Wang, Yuxiang; Lu, Shijin; Li, Jingsong; Yin, Xinhua; Guo, Qiang

    2014-01-01

    The objective of the study is to explore the possible association of the monocyte chemoattractant protein (MCP)-1-362G/C genetic polymorphism and plasma levels of MCP-1 in patients with spinal tuberculosis (TB). The MCP-1-362G/C (rs2857656) polymorphism and blood levels of MCP-1 in patients with spinal TB and healthy subjects were evaluated and compared. Three hundred thirty-two patients and 336 healthy subjects were genotyped using polymerase chain reaction and Sanger DNA sequencing technology. MCP-1 plasma levels were measured by a solid-phase enzyme-linked immunosorbent assay. When comparisons were made between patients and controls, the frequency of the MCP-1-362*C minor allele (55.4% versus 47.5%, P = 0.004, odds ratio [OR] = 1.376, 95% confidence interval [CI]: 1.109-1.706) and the carriers of the MCP-1-362*C allele (80.7% versus 71.4%, P = 0.005, OR = 1. 657, 95% CI: 1.167-2.403) were over-represented in patients. The mean MCP-1 plasma level in spinal TB patients was significantly higher than in controls (154.44 ± 68.81 pg/mL versus 36.69 ± 21.71 pg/mL, t = -5.85, P < 0.001). The patients with the CC genotype had the highest MCP-1 level (150.63 ± 73.89 pg/mL), followed by those with the GC genotype (108.63 ± 52.09 pg/mL, t = 2.351, P = 0.022) and GG (91.29 ± 54.31 pg/mL, t = 3.091, P = 0.003) homozygotes. We report the association of the -362G/C genetic polymorphism and increased plasma levels of MCP-1 in patients with spinal TB and nominate the -362*C minor allele as a risk factor for spinal TB in the Chinese population.

  3. Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

    Directory of Open Access Journals (Sweden)

    Aurélie Fougère

    2016-11-01

    Full Text Available Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC. P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene families are exported onto the surface of infected red blood cells (iRBC and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understand the role of RBC-exported proteins of rodent malaria parasites (RMP we analysed the expression and cellular location by fluorescent-tagging of members of the pir, fam-a and fam-b multigene families. Furthermore, we performed phylogenetic analyses of the fam-a and fam-b multigene families, which indicate that both families have a history of functional differentiation unique to RMP. We demonstrate for all three families that expression of family members in iRBC is not mutually exclusive. Most tagged proteins were transported into the iRBC cytoplasm but not onto the iRBC plasma membrane, indicating that they are unlikely to play a direct role in iRBC-host cell interactions. Unexpectedly, most family members are also expressed during the liver stage, where they are transported into the parasitophorous vacuole. This suggests that these protein families promote parasite development in both the liver and blood, either by supporting parasite development within hepatocytes and erythrocytes and/or by manipulating the host immune response. Indeed, in the case of Fam-A, which have a steroidogenic acute regulatory-related lipid transfer (START domain, we found that several family members can transfer phosphatidylcholine in vitro. These observations indicate that these proteins may transport (host phosphatidylcholine for membrane synthesis. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites.

  4. Relationship between a common variant in the fatty acid desaturase (FADS) cluster and eicosanoid generation in humans.

    Science.gov (United States)

    Hester, Austin G; Murphy, Robert C; Uhlson, Charis J; Ivester, Priscilla; Lee, Tammy C; Sergeant, Susan; Miller, Leslie R; Howard, Timothy D; Mathias, Rasika A; Chilton, Floyd H

    2014-08-08

    Dramatic shifts in the Western diet have led to a marked increase in the dietary intake of the n-6 polyunsaturated fatty acid (PUFA), linoleic acid (LA). Dietary LA can then be converted to arachidonic acid (ARA) utilizing three enzymatic steps. Two of these steps are encoded for by the fatty acid desaturase (FADS) cluster (chromosome 11, 11q12.2-q13) and certain genetic variants within the cluster are highly associated with ARA levels. However, no study to date has examined whether these variants further influence pro-inflammatory, cyclooxygenase and lipoxygenase eicosanoid products. This study examined the impact of a highly influential FADS SNP, rs174537 on leukotriene, HETE, prostaglandin, and thromboxane biosynthesis in stimulated whole blood. Thirty subjects were genotyped at rs174537 (GG, n = 11; GT, n = 13; TT, n = 6), a panel of fatty acids from whole serum was analyzed, and precursor-to-product PUFA ratios were calculated as a marker of the capacity of tissues (particularly the liver) to synthesize long chain PUFAs. Eicosanoids produced by stimulated human blood were measured by LC-MS/MS. We observed an association between rs174537 and the ratio of ARA/LA, leukotriene B4, and 5-HETE but no effect on levels of cyclooxygenase products. Our results suggest that variation at rs174537 not only impacts the synthesis of ARA but the overall capacity of whole blood to synthesize 5-lipoxygenase products; these genotype-related changes in eicosanoid levels could have important implications in a variety of inflammatory diseases.

  5. The BRCA1 variant p.Ser36Tyr abrogates BRCA1 protein function and potentially confers a moderate risk of breast cancer.

    Science.gov (United States)

    Christou, Charita M; Hadjisavvas, Andreas; Kyratzi, Maria; Flouri, Christina; Neophytou, Ioanna; Anastasiadou, Violetta; Loizidou, Maria A; Kyriacou, Kyriacos

    2014-01-01

    The identification of variants of unknown clinical significance (VUS) in the BRCA1 gene complicates genetic counselling and causes additional anxiety to carriers. In silico approaches currently used for VUS pathogenicity assessment are predictive and often produce conflicting data. Furthermore, functional assays are either domain or function specific, thus they do not examine the entire spectrum of BRCA1 functions and interpretation of individual assay results can be misleading. PolyPhen algorithm predicted that the BRCA1 p.Ser36Tyr VUS identified in the Cypriot population was damaging, whereas Align-GVGD predicted that it was possibly of no significance. In addition the BRCA1 p.Ser36Tyr variant was found to be associated with increased risk (OR = 3.47, 95% CI 1.13-10.67, P = 0.02) in a single case-control series of 1174 cases and 1109 controls. We describe a cellular system for examining the function of exogenous full-length BRCA1 and for classifying VUS. We achieved strong protein expression of full-length BRCA1 in transiently transfected HEK293T cells. The p.Ser36Tyr VUS exhibited low protein expression similar to the known pathogenic variant p.Cys61Gly. Co-precipitation analysis further demonstrated that it has a reduced ability to interact with BARD1. Further, co-precipitation analysis of nuclear and cytosolic extracts as well as immunofluorescence studies showed that a high proportion of the p.Ser36Tyr variant is withheld in the cytoplasm contrary to wild type protein. In addition the ability of p.Ser36Tyr to co-localize with conjugated ubiquitin foci in the nuclei of S-phase synchronized cells following genotoxic stress with hydroxyurea is impaired at more pronounced levels than that of the p.Cys61Gly pathogenic variant. The p.Ser36Tyr variant demonstrates abrogated function, and based on epidemiological, genetic, and clinical data we conclude that the p.Ser36Tyr variant is probably associated with a moderate breast cancer risk.

  6. NAPPA as a Real New Method for Protein Microarray Generation.

    Science.gov (United States)

    Díez, Paula; González-González, María; Lourido, Lucía; Dégano, Rosa M; Ibarrola, Nieves; Casado-Vela, Juan; LaBaer, Joshua; Fuentes, Manuel

    2015-04-24

    Nucleic Acid Programmable Protein Arrays (NAPPA) have emerged as a powerful and innovative technology for the screening of biomarkers and the study of protein-protein interactions, among others possible applications. The principal advantages are the high specificity and sensitivity that this platform offers. Moreover, compared to conventional protein microarrays, NAPPA technology avoids the necessity of protein purification, which is expensive and time-consuming, by substituting expression in situ with an in vitro transcription/translation kit. In summary, NAPPA arrays have been broadly employed in different studies improving knowledge about diseases and responses to treatments. Here, we review the principal advances and applications performed using this platform during the last years.

  7. Data on the evolutionary history of the V(DJ recombination-activating protein 1 – RAG1 coupled with sequence and variant analyses

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    Abhishek Kumar

    2016-09-01

    Full Text Available RAG1 protein is one of the key component of RAG complex regulating the V(DJ recombination. There are only few studies for RAG1 concerning evolutionary history, detailed sequence and mutational hotspots. Herein, we present out datasets used for the recent comprehensive study of RAG1 based on sequence, phylogenetic and genetic variant analyses (Kumar et al., 2015 [1]. Protein sequence alignment helped in characterizing the conserved domains and regions of RAG1. It also aided in unraveling ancestral RAG1 in the sea urchin. Human genetic variant analyses revealed 751 mutational hotspots, located both in the coding and the non-coding regions. For further analysis and discussion, see (Kumar et al., 2015 [1].

  8. Generation of high-performance binding proteins for peptide motifs by affinity clamping

    OpenAIRE

    Koide, Shohei; Huang, Jin

    2013-01-01

    We describe concepts and methodologies for generating “Affinity Clamps”, a new class of recombinant binding proteins that achieve high affinity and high specificity toward short peptide motifs of biological importance, which is a major challenge in protein engineering. The Affinity Clamping concept exploits the potential of nonhomologous recombination of protein domains in generating large changes in protein function and the inherent binding affinity and specificity of the so-called modular i...

  9. Common genetic variants of surfactant protein-D (SP-D are associated with type 2 diabetes.

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    Neus Pueyo

    Full Text Available CONTEXT: Surfactant protein-D (SP-D is a primordial component of the innate immune system intrinsically linked to metabolic pathways. We aimed to study the association of single nucleotide polymorphisms (SNPs affecting SP-D with insulin resistance and type 2 diabetes (T2D. RESEARCH DESIGN AND METHODS: We evaluated a common genetic variant located in the SP-D coding region (rs721917, Met(31Thr in a sample of T2D patients and non-diabetic controls (n = 2,711. In a subset of subjects (n = 1,062, this SNP was analyzed in association with circulating SP-D concentrations, insulin resistance, and T2D. This SNP and others were also screened in the publicly available Genome Wide Association (GWA database of the Meta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC. RESULTS: We found the significant association of rs721917 with circulating SP-D, parameters of insulin resistance and T2D. Indeed, G carriers showed decreased circulating SP-D (p = 0.004, decreased fasting glucose (p = 0.0002, glycated hemoglobin (p = 0.0005, and 33% (p = 0.002 lower prevalence of T2D, estimated under a dominant model, especially among women. Interestingly, these differences remained significant after controlling for origin, age, gender, and circulating SP-D. Moreover, this SNP and others within the SP-D genomic region (i.e. rs10887344 were significantly associated with quantitative measures of glucose homeostasis, insulin sensitivity, and T2D, according to GWAS datasets from MAGIC. CONCLUSIONS: SP-D gene polymorphisms are associated with insulin resistance and T2D. These associations are independent of circulating SP-D concentrations.

  10. Multiple length peptide-pheromone variants produced by Streptococcus pyogenes directly bind Rgg proteins to confer transcriptional regulation.

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    Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Nanavati, Dhaval; Federle, Michael J

    2014-08-08

    Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nm and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication.

  11. Coexistence of sense and anti-sense mRNAs of variant surface protein in Giardia lamblia trophozoites.

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    Guo, Junli; Zheng, Wenyu; Wang, Yuehua; Li, Yao; Lu, Siqi; Feng, Xianmin

    2014-02-14

    A strategy of the parasitic protozoan Giardia lamblia to evade attack from the host immune system is periodic changes of its surface antigen, a member of the variant surface protein (VSP) family. A post-transcriptional gene silencing mechanism has been proposed to explain the presence of only one among many possible VSPs at any time. To investigate this phenomenon further, we extracted total RNA from cultured trophozoites of the G. lamblia C2 isolate, and cDNA was reverse-transcribed from the RNA. Sense and anti-sense VSPs were amplified from the total cDNA using nested PCR with primers designed from the 3'-conserved region and the known 5' or 3' end of the cDNA library. Sequence analyses of the amplified products revealed more than 34 full-length antisense VSPs and a smear of sense VSPs. Sequence alignments and comparisons revealed that these VSPs contained variable N-termini and conserved C-termini, and could be classified into 5 clades based on the sizes and variations of the N-terminal sequence. All antisense VSPs existed in the sense forms, but no corresponding antisense VSP existed for sense RNA (snsRNA) 16. The coexistence of sense and antisense VSP mRNAs in cultured G. lamblia supports the post-transcriptional regulation of VSP expression. We propose that VSPs transcribed simultaneously in the sense and antisense forms form double-stranded RNAs (dsRNAs) which are degraded by the Dicer endonuclease, while a VSP without an antisense transcription (e.g., snsRNA16) will be expressed on the surface of Giardia. In addition, in the course of this investigation VSPs were identified that were previously not known. PCR-based amplification of specific sense and antisense VSP cDNAs can be used to identify the specific VSP on G. lamblia trophozoites, which is easier than using specific monoclonal antibody approaches.

  12. SDS, a structural disruption score for assessment of missense variant deleteriousness

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    Thanawadee ePreeprem

    2014-04-01

    Full Text Available We have developed a novel structure-based evaluation for missense variants that explicitly models protein structure and amino acid properties to predict the likelihood that a variant disrupts protein function. A structural disruption score (SDS is introduced as a measure to depict the likelihood that a case variant is functional. The score is constructed using characteristics that distinguish between causal and neutral variants within a group of proteins. The SDS score is correlated with standard sequence-based deleteriousness, but shows promise for improving discrimination between neutral and causal variants at less conserved sites.The prediction was performed on 3-dimentional structures of 57 gene products whose homozygous SNPs were identified as case-exclusive variants in an exome sequencing study of epilepsy disorders. We contrasted the candidate epilepsy variants with scores for likely benign variants found in the EVS database, and for positive control variants in the same genes that are suspected to promote a range of diseases. To derive a characteristic profile of damaging SNPs, we transformed continuous scores into categorical variables based on the score distribution of each measurement, collected from all possible SNPs in this protein set, where extreme measures were assumed to be deleterious. A second epilepsy dataset was used to replicate the findings. Causal variants tend to receive higher sequence-based deleterious scores, induce larger physico-chemical changes between amino acid pairs, locate in protein domains, buried sites or on conserved protein surface clusters, and cause protein destabilization, relative to negative controls. These measures were agglomerated for each variant. A list of nine high-priority putative functional variants for epilepsy was generated. Our newly developed SDS protocol facilitates SNP prioritization for experimental validation.

  13. Molecular dynamics and docking simulation of a natural variant of Activated Protein C with impaired protease activity: implications for integrin-mediated antiseptic function.

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    D'Ursi, Pasqualina; Orro, Alessandro; Morra, Giulia; Moscatelli, Marco; Trombetti, Gabriele; Milanesi, Luciano; Rovida, Ermanna

    2015-01-01

    Activated Protein C (APC) is a multifunctional serine protease, primarily known for its anticoagulant function in the coagulation system. Several studies have already elucidated its role in counteracting apoptosis and inflammation in cells, while significant effort is still ongoing for defining its involvement in sepsis. Earlier literature has shown that the antiseptic function of APC is mediated by its binding to leukocyte integrins, which is due to the presence of the integrin binding motif Arg-Gly-Asp at the N-terminus of the APC catalytic chain. Many natural mutants have been identified in patients with Protein C deficiency diagnosis including a variant of specificity pocket (Gly216Asp). In this work, we present a molecular model of the complex of APC with αVβ3 integrin obtained by protein-protein docking approach. A computational analysis of this variant is hereby presented, based on molecular dynamics and docking simulations, aiming at investigating the effects of the Gly216Asp mutation on the protein conformation and inferring its functional implications. Our study shows that such mutation is likely to impair the protease activity while preserving the overall protein fold. Moreover, superposition of the integrin binding motifs in wild-type and mutant forms suggests that the interaction with integrin can still occur and thus the mutant is likely to retain its antiseptic function related to the neutrophyl integrin binding. Therapeutic applications could result in this APC mutant which retains antiseptic function without anticoagulant side effects.

  14. The Staphylococcus aureus NuoL-like protein MpsA contributes to the generation of membrane potential.

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    Mayer, Sonja; Steffen, Wojtek; Steuber, Julia; Götz, Friedrich

    2015-03-01

    In aerobic microorganisms, the entry point of respiratory electron transfer is represented by the NADH:quinone oxidoreductase. The enzyme couples the oxidation of NADH with the reduction of quinone. In the type 1 NADH:quinone oxidoreductase (Ndh1), this reaction is accompanied by the translocation of cations, such as H(+) or Na(+). In Escherichia coli, cation translocation is accomplished by the subunit NuoL, thus generating membrane potential (Δψ). Some microorganisms achieve NADH oxidation by the alternative, nonelectrogenic type 2 NADH:quinone oxidoreductase (Ndh2), which is not cation translocating. Since these enzymes had not been described in Staphylococcus aureus, the goal of this study was to identify proteins operating in the NADH:quinone segment of its respiratory chain. We demonstrated that Ndh2 represents a NADH:quinone oxidoreductase in S. aureus. Additionally, we identified a hypothetical protein in S. aureus showing sequence similarity to the proton-translocating subunit NuoL of complex I in E. coli: the NuoL-like protein MpsA. Mutants with deletion of the nuoL-like gene mpsA and its corresponding operon, mpsABC (mps for membrane potential-generating system), exhibited a small-colony-variant-like phenotype and were severely affected in Δψ and oxygen consumption rates. The MpsABC proteins did not confer NADH oxidation activity. Using an Na(+)/H(+) antiporter-deficient E. coli strain, we could show that MpsABC constitute a cation-translocating system capable of Na(+) transport. Our study demonstrates that MpsABC represent an important functional system of the respiratory chain of S. aureus that acts as an electrogenic unit responsible for the generation of Δψ.

  15. Rapid and Highly Sensitive Detection of Variant Creutzfeldt-Jakob Disease Abnormal Prion Protein on Steel Surfaces by Protein Misfolding Cyclic Amplification: Application to Prion Decontamination Studies.

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    Maxime Belondrade

    Full Text Available The prevalence of variant Creutzfeldt-Jakob disease (vCJD in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA. This assay allowed the specific detection of minute quantities (10-8 brain dilution of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.

  16. Rapid and Highly Sensitive Detection of Variant Creutzfeldt-Jakob Disease Abnormal Prion Protein on Steel Surfaces by Protein Misfolding Cyclic Amplification: Application to Prion Decontamination Studies.

    Science.gov (United States)

    Belondrade, Maxime; Nicot, Simon; Béringue, Vincent; Coste, Joliette; Lehmann, Sylvain; Bougard, Daisy

    2016-01-01

    The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10-8 brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.

  17. Genetic Variance in Uncoupling Protein 2 in Relation to Obesity, Type 2 Diabetes, and Related Metabolic Traits: Focus on the Functional −866G>A Promoter Variant (rs659366

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    Louise T. Dalgaard

    2011-01-01

    Full Text Available Uncoupling proteins (UCPs are mitochondrial proteins able to dissipate the proton gradient of the inner mitochondrial membrane when activated. This decreases ATP-generation through oxidation of fuels and may theoretically decrease energy expenditure leading to obesity. Evidence from Ucp(−/− mice revealed a role of UCP2 in the pancreatic β-cell, because β-cells without UCP2 had increased glucose-stimulated insulin secretion. Thus, from being a candidate gene for obesity UCP2 became a valid candidate gene for type 2 diabetes mellitus. This prompted a series of studies of the human UCP2 and UCP3 genes with respect to obesity and diabetes. Of special interest was a promoter variant of UCP2 situated 866bp upstream of transcription initiation (−866G>A, rs659366. This variant changes promoter activity and has been associated with obesity and/or type 2 diabetes in several, although not all, studies. The aim of the current paper is to summarize current evidence of association of UCP2 genetic variation with obesity and type 2 diabetes, with focus on the −866G>A polymorphism.

  18. The microRNAs in an ancient protist repress the variant-specific surface protein expression by targeting the entire coding sequence.

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    Ashesh A Saraiya

    2014-02-01

    Full Text Available microRNAs (miRNA have been detected in the deeply branched protist, Giardia lamblia, and shown to repress expression of the family of variant-specific surface proteins (VSPs, only one of which is expressed in Giardia trophozoite at a given time. Three next-generation sequencing libraries of Giardia Argonaute-associated small RNAs were constructed and analyzed. Analysis of the libraries identified a total of 99 new putative miRNAs with a size primarily in the 26 nt range similar to the size previously predicted by the Giardia Dicer crystal structure and identified by our own studies. Bioinformatic analysis identified multiple putative miRNA target sites in the mRNAs of all 73 VSPs. The effect of miRNA target sites within a defined 3'-region were tested on two vsp mRNAs. All the miRNAs showed partial repression of the corresponding vsp expression and were additive when the targeting sites were separately located. But the combined repression still falls short of 100%. Two other relatively short vsp mRNAs with 15 and 11 putative miRNA target sites identified throughout their ORFs were tested with their corresponding miRNAs. The results indicate that; (1 near 100% repression of vsp mRNA expression can be achieved through the combined action of multiple miRNAs on target sites located throughout the ORF; (2 the miRNA machinery could be instrumental in repressing the expression of vsp genes in Giardia; (3 this is the first time that all the miRNA target sites in the entire ORF of a mRNA have been tested and shown to be functional.

  19. NAPPA as a Real New Method for Protein Microarray Generation

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    Paula Díez

    2015-04-01

    Full Text Available Nucleic Acid Programmable Protein Arrays (NAPPA have emerged as a powerful and innovative technology for the screening of biomarkers and the study of protein-protein interactions, among others possible applications. The principal advantages are the high specificity and sensitivity that this platform offers. Moreover, compared to conventional protein microarrays, NAPPA technology avoids the necessity of protein purification, which is expensive and time-consuming, by substituting expression in situ with an in vitro transcription/translation kit. In summary, NAPPA arrays have been broadly employed in different studies improving knowledge about diseases and responses to treatments. Here, we review the principal advances and applications performed using this platform during the last years.

  20. A viable simian virus 40 variant with a deletion in the overlapping genes for virion proteins VP1, VP2 and VP3.

    Science.gov (United States)

    Norkin, L C; Piatak, M

    1982-12-01

    Nucleotide sequence analysis was used to determine the exact location of a deletion in the late region of the SP2 mutant of simian virus 40 (SV40), a viable small-plaque variant isolated from a persistent infection of rhesus monkey kidney cells. The results indicate that six base pairs are deleted from that part of the SV40 genome in which the coding regions for the three virion proteins, VP1, VP2 and VP3, overlap. This implies that all three virion proteins are affected by the deletion. This finding is discussed with respect to the viability of SP2.

  1. Structural and functional interaction of fatty acids with human liver fatty acid-binding protein (L-FABP) T94A variant.

    Science.gov (United States)

    Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Landrock, Kerstin K; Landrock, Danilo; Gupta, Shipra; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

    2014-05-01

    The human liver fatty acid-binding protein (L-FABP) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride levels. How this amino acid substitution elicits these effects is not known. This issue was addressed using human recombinant wild-type (WT) and T94A variant L-FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT, TC and CC). The T94A substitution did not alter or only slightly altered L-FABP binding affinities for saturated, monounsaturated or polyunsaturated long chain fatty acids, nor did it change the affinity for intermediates of triglyceride synthesis. Nevertheless, the T94A substitution markedly altered the secondary structural response of L-FABP induced by binding long chain fatty acids or intermediates of triglyceride synthesis. Finally, the T94A substitution markedly decreased the levels of induction of peroxisome proliferator-activated receptor α-regulated proteins such as L-FABP, fatty acid transport protein 5 and peroxisome proliferator-activated receptor α itself meditated by the polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid in cultured primary human hepatocytes. Thus, although the T94A substitution did not alter the affinity of human L-FABP for long chain fatty acids, it significantly altered human L-FABP structure and stability, as well as the conformational and functional response to these ligands.

  2. The contribution of a 9p21.3 variant, a KIF6 variant, and C-reactive protein to predicting risk of myocardial infarction in a prospective study

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    Tracy Russell P

    2011-03-01

    Full Text Available Abstract Background Genetic risk factors might improve prediction of coronary events. Several variants at chromosome 9p21.3 have been widely reported to be associated with coronary heart disease (CHD in prospective and case-control studies. A variant of KIF6 (719Arg has also been reported to be associated with increased risk of CHD in large prospective studies, but not in case-control studies. We asked whether the addition of genetic information (the 9p21.3 or KIF6 variants or a well-established non-genetic risk factor (C-reactive protein [CRP] can improve risk prediction by the Framingham Risk Score (FRS in the Cardiovascular Health Study (CHS--a prospective observational study of risk factors for cardiovascular disease among > 5,000 participants aged 65 or older. Methods Improvement of risk prediction was assessed by change in the area under the receiver-operator characteristic curve (AUC and by net reclassification improvement (NRI. Results Among white participants the FRS was improved by addition of KIF6 719Arg carrier status among men as assessed by the AUC (from 0.581 to 0.596, P = 0.03 but not by NRI (NRI = 0.027, P = 0.32. Adding both CRP and 719Arg carrier status to the FRS improved risk prediction by the AUC (0.608, P = 0.02 and NRI (0.093, P = 0.008 in men, but not women (P ≥ 0.24. Conclusions While none of these risk markers individually or in combination improved risk prediction among women, a combination of KIF6 719Arg carrier status and CRP levels modestly improved risk prediction among white men; although this improvement is not significant after multiple-testing correction. These observations should be investigated in other prospective studies.

  3. Did Convergent Protein Evolution Enable Phytoplasmas to Generate 'Zombie Plants'?

    Science.gov (United States)

    Rümpler, Florian; Gramzow, Lydia; Theißen, Günter; Melzer, Rainer

    2015-12-01

    Phytoplasmas are pathogenic bacteria that reprogram plant development such that leaf-like structures instead of floral organs develop. Infected plants are sterile and mainly serve to propagate phytoplasmas and thus have been termed 'zombie plants'. The developmental reprogramming relies on specific interactions of the phytoplasma protein SAP54 with a small subset of MADS-domain transcription factors. Here, we propose that SAP54 folds into a structure that is similar to that of the K-domain, a protein-protein interaction domain of MADS-domain proteins. We suggest that undergoing convergent structural and sequence evolution, SAP54 evolved to mimic the K-domain. Given the high specificity of resulting developmental alterations, phytoplasmas might be used to study flower development in genetically intractable plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. G protein coupled receptors as targets for next generation pesticides.

    Science.gov (United States)

    Audsley, Neil; Down, Rachel E

    2015-12-01

    There is an on-going need for the discovery and development of new pesticides due to the loss of existing products through the continuing development of resistance, the desire for products with more favourable environmental and toxicological profiles and the need to implement the principles of integrated pest management. Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behaviour, including reproduction, osmoregulation, growth and development. Modifying normal receptor function by blocking or over stimulating its actions may either result in the death of a pest or disrupt its normal fitness or reproductive capacity to reduce pest populations. Hence GPCRs offer potential targets for the development of next generation pesticides providing opportunities to discover new chemistries for invertebrate pest control. Such receptors are important targets for pharmaceutical drugs, but are under-exploited by the agro-chemical industry. The octopamine receptor agonists are the only pesticides with a recognized mode of action, as described in the classification scheme developed by the Insecticide Resistance Action Committee, that act via a GPCR. The availability of sequenced insect genomes has facilitated the characterization of insect GPCRs, but the development and utilization of screening assays to identify lead compounds has been slow. Various studies using knock-down technologies or applying the native ligands and/or neuropeptide analogues to pest insects in vivo, have however demonstrated that modifying normal receptor function can have an insecticidal effect. This review presents examples of potential insect neuropeptide receptors that are potential targets for lead compound development, using case studies from three representative pest species, Tribolium castaneum, Acyrthosiphon pisum, and Drosophila suzukii. Functional analysis studies on T. castaneum suggest that GPCRs involved in growth and development (eclosion

  5. Truncated G protein-coupled mu opioid receptor MOR-1 splice variants are targets for highly potent opioid analgesics lacking side effects.

    Science.gov (United States)

    Majumdar, Susruta; Grinnell, Steven; Le Rouzic, Valerie; Burgman, Maxim; Polikar, Lisa; Ansonoff, Michael; Pintar, John; Pan, Ying-Xian; Pasternak, Gavril W

    2011-12-06

    Pain remains a pervasive problem throughout medicine, transcending all specialty boundaries. Despite the extraordinary insights into pain and its mechanisms over the past few decades, few advances have been made with analgesics. Most pain remains treated by opiates, which have significant side effects that limit their utility. We now describe a potent opiate analgesic lacking the traditional side effects associated with classical opiates, including respiratory depression, significant constipation, physical dependence, and, perhaps most important, reinforcing behavior, demonstrating that it is possible to dissociate side effects from analgesia. Evidence indicates that this agent acts through a truncated, six-transmembrane variant of the G protein-coupled mu opioid receptor MOR-1. Although truncated splice variants have been reported for a number of G protein-coupled receptors, their functional relevance has been unclear. Our evidence now suggests that truncated variants can be physiologically important through heterodimerization, even when inactive alone, and can comprise new therapeutic targets, as illustrated by our unique opioid analgesics with a vastly improved pharmacological profile.

  6. TYK2 Protein-Coding Variants Protect against Rheumatoid Arthritis and Autoimmunity, with No Evidence of Major Pleiotropic Effects on Non-Autoimmune Complex Traits

    Science.gov (United States)

    Diogo, Dorothée; Bastarache, Lisa; Liao, Katherine P.; Graham, Robert R.; Fulton, Robert S.; Greenberg, Jeffrey D.; Eyre, Steve; Bowes, John; Cui, Jing; Lee, Annette; Pappas, Dimitrios A.; Kremer, Joel M.; Barton, Anne; Coenen, Marieke J. H.; Franke, Barbara; Kiemeney, Lambertus A.; Mariette, Xavier; Richard-Miceli, Corrine; Canhão, Helena; Fonseca, João E.; de Vries, Niek; Tak, Paul P.; Crusius, J. Bart A.; Nurmohamed, Michael T.; Kurreeman, Fina; Mikuls, Ted R.; Okada, Yukinori; Stahl, Eli A.; Larson, David E.; Deluca, Tracie L.; O'Laughlin, Michelle; Fronick, Catrina C.; Fulton, Lucinda L.; Kosoy, Roman; Ransom, Michael; Bhangale, Tushar R.; Ortmann, Ward; Cagan, Andrew; Gainer, Vivian; Karlson, Elizabeth W.; Kohane, Isaac; Murphy, Shawn N.; Martin, Javier; Zhernakova, Alexandra; Klareskog, Lars; Padyukov, Leonid; Worthington, Jane; Mardis, Elaine R.; Seldin, Michael F.; Gregersen, Peter K.; Behrens, Timothy; Raychaudhuri, Soumya; Denny, Joshua C.; Plenge, Robert M.

    2015-01-01

    Despite the success of genome-wide association studies (GWAS) in detecting a large number of loci for complex phenotypes such as rheumatoid arthritis (RA) susceptibility, the lack of information on the causal genes leaves important challenges to interpret GWAS results in the context of the disease biology. Here, we genetically fine-map the RA risk locus at 19p13 to define causal variants, and explore the pleiotropic effects of these same variants in other complex traits. First, we combined Immunochip dense genotyping (n = 23,092 case/control samples), Exomechip genotyping (n = 18,409 case/control samples) and targeted exon-sequencing (n = 2,236 case/controls samples) to demonstrate that three protein-coding variants in TYK2 (tyrosine kinase 2) independently protect against RA: P1104A (rs34536443, OR = 0.66, P = 2.3x10-21), A928V (rs35018800, OR = 0.53, P = 1.2x10-9), and I684S (rs12720356, OR = 0.86, P = 4.6x10-7). Second, we show that the same three TYK2 variants protect against systemic lupus erythematosus (SLE, Pomnibus = 6x10-18), and provide suggestive evidence that two of the TYK2 variants (P1104A and A928V) may also protect against inflammatory bowel disease (IBD; Pomnibus = 0.005). Finally, in a phenome-wide association study (PheWAS) assessing >500 phenotypes using electronic medical records (EMR) in >29,000 subjects, we found no convincing evidence for association of P1104A and A928V with complex phenotypes other than autoimmune diseases such as RA, SLE and IBD. Together, our results demonstrate the role of TYK2 in the pathogenesis of RA, SLE and IBD, and provide supporting evidence for TYK2 as a promising drug target for the treatment of autoimmune diseases. PMID:25849893

  7. TYK2 protein-coding variants protect against rheumatoid arthritis and autoimmunity, with no evidence of major pleiotropic effects on non-autoimmune complex traits.

    Directory of Open Access Journals (Sweden)

    Dorothée Diogo

    Full Text Available Despite the success of genome-wide association studies (GWAS in detecting a large number of loci for complex phenotypes such as rheumatoid arthritis (RA susceptibility, the lack of information on the causal genes leaves important challenges to interpret GWAS results in the context of the disease biology. Here, we genetically fine-map the RA risk locus at 19p13 to define causal variants, and explore the pleiotropic effects of these same variants in other complex traits. First, we combined Immunochip dense genotyping (n = 23,092 case/control samples, Exomechip genotyping (n = 18,409 case/control samples and targeted exon-sequencing (n = 2,236 case/controls samples to demonstrate that three protein-coding variants in TYK2 (tyrosine kinase 2 independently protect against RA: P1104A (rs34536443, OR = 0.66, P = 2.3 x 10(-21, A928V (rs35018800, OR = 0.53, P = 1.2 x 10(-9, and I684S (rs12720356, OR = 0.86, P = 4.6 x 10(-7. Second, we show that the same three TYK2 variants protect against systemic lupus erythematosus (SLE, Pomnibus = 6 x 10(-18, and provide suggestive evidence that two of the TYK2 variants (P1104A and A928V may also protect against inflammatory bowel disease (IBD; P(omnibus = 0.005. Finally, in a phenome-wide association study (PheWAS assessing >500 phenotypes using electronic medical records (EMR in >29,000 subjects, we found no convincing evidence for association of P1104A and A928V with complex phenotypes other than autoimmune diseases such as RA, SLE and IBD. Together, our results demonstrate the role of TYK2 in the pathogenesis of RA, SLE and IBD, and provide supporting evidence for TYK2 as a promising drug target for the treatment of autoimmune diseases.

  8. Cellulase variants

    Energy Technology Data Exchange (ETDEWEB)

    Blazej, Robert; Toriello, Nicholas; Emrich, Charles; Cohen, Richard N.; Koppel, Nitzan

    2015-07-14

    This invention provides novel variant cellulolytic enzymes having improved activity and/or stability. In certain embodiments the variant cellulotyic enzymes comprise a glycoside hydrolase with or comprising a substitution at one or more positions corresponding to one or more of residues F64, A226, and/or E246 in Thermobifida fusca Cel9A enzyme. In certain embodiments the glycoside hydrolase is a variant of a family 9 glycoside hydrolase. In certain embodiments the glycoside hydrolase is a variant of a theme B family 9 glycoside hydrolase.

  9. Characterization of variant Creutzfeldt-Jakob disease prions in prion protein-humanized mice carrying distinct codon 129 genotypes.

    Science.gov (United States)

    Takeuchi, Atsuko; Kobayashi, Atsushi; Ironside, James W; Mohri, Shirou; Kitamoto, Tetsuyuki

    2013-07-26

    To date, all clinical variant Creutzfeldt-Jakob disease (vCJD) patients are homozygous for methionine at polymorphic codon 129 (129M/M) of the prion protein (PrP) gene. However, the appearance of asymptomatic secondary vCJD infection in individuals with a PRNP codon 129 genotype other than M/M and transmission studies using animal models have raised the concern that all humans might be susceptible to vCJD prions, especially via secondary infection. To reevaluate this possibility and to analyze in detail the transmission properties of vCJD prions to transgenic animals carrying distinct codon 129 genotype, we performed intracerebral inoculation of vCJD prions to humanized knock-in mice carrying all possible codon 129 genotypes (129M/M, 129M/V, or 129V/V). All humanized knock-in mouse lines were susceptible to vCJD infection, although the attack rate gradually decreased from 129M/M to 129M/V and to 129V/V. The amount of PrP deposition including florid/amyloid plaques in the brain also gradually decreased from 129M/M to 129M/V and to 129V/V. The biochemical properties of protease-resistant abnormal PrP in the brain and transmissibility of these humanized mouse-passaged vCJD prions upon subpassage into knock-in mice expressing bovine PrP were not affected by the codon 129 genotype. These results indicate that individuals with the 129V/V genotype may be more susceptible to secondary vCJD infection than expected and may lack the neuropathological characteristics observed in vCJD patients with the 129M/M genotype. Besides the molecular typing of protease-resistant PrP in the brain, transmission studies using knock-in mice carrying bovine PrP may aid the differential diagnosis of secondary vCJD infection, especially in individuals with the 129V/V genotype.

  10. The profile of snoRNA-derived microRNAs that regulate expression of variant surface proteins in Giardia lamblia.

    Science.gov (United States)

    Li, Wei; Saraiya, Ashesh A; Wang, Ching C

    2012-09-01

    In the current investigation, we analysed all the known small nucleolar RNAs (snoRNAs) in the deeply branching protozoan parasite Giardia lamblia for potential microRNAs (miRNAs) that might be derived from them. Two putative miRNAs have since been identified by Northern blot, primer extension, 3' RACE and co-immunoprecipitation with Giardia Argonaute (GlAgo), and designated miR6 and miR10. Giardia Dicer (GlDcr) is capable of processing the snoRNAs into the corresponding miRNAs in vitro. Potential miR6 and miR10 binding sites in Giardia genome were predicted bio-informatically. A miR6 binding site was found at the 3' untranslated regions (UTR) of 44 variant surface protein (vsp) genes, whereas a miR10 binding site was identified at the 3' end of 159 vsp open-reading frames. Thirty-three of these vsp genes turned out to contain binding sites for both miR6 and miR10. A reporter mRNA tagged with the 3' end of vsp1267, which contains the target sites for both miRNAs, was translationally repressed by both miRNAs in Giardia. Episomal expression of an N-terminal c-myc tagged VSP1267 was found significantly repressed by introducing either miR6 or miR10 into the cells and the repressive effects were additive. When the 2'-O-methyl antisense oligos (ASOs) of either miR6 or miR10 was introduced, however, there was an enhancement of tagged VSP1267 expression suggesting an inhibition of the repressive effects of endogenous miR6 or miR10 by the ASOs. Of the total 220 vsp genes in Giardia, we have now found 178 of them carrying putative binding sites for all the miRNAs that have been currently identified, suggesting that miRNAs are likely the regulators of VSP expression in Giardia.

  11. Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

    OpenAIRE

    Li, Wuping; Zhang, Liqun; Johnson, Jarrod S; Zhijian, Wu; Grieger, Joshua C; Ping-Jie, Xiao; Drouin, Lauren M; Agbandje-McKenna, Mavis; Pickles, Raymond J.; Samulski, R. Jude

    2009-01-01

    Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an...

  12. HUMAN LIVER FATTY ACID BINDING PROTEIN (L-FABP) T94A VARIANT ALTERS STRUCTURE, STABILITY, AND INTERACTION WITH FIBRATES

    OpenAIRE

    Martin, Gregory G.; McIntosh, Avery L.; Huang, Huan; Gupta, Shipra; Atshaves, Barbara P.; Landrock, Kerstin K.; Landrock, Danilo; Kier, Ann B.; Schroeder, Friedhelm

    2013-01-01

    Although the human L-FABP T94A variant arises from the most commonly occurring SNP in the entire FABP family, there is a complete lack of understanding regarding the role of this polymorphism in human disease. It has been hypothesized that the T94A substitution results in complete loss of ligand binding ability and function analogous to L-FABP gene ablation. This possibility was addressed using recombinant human WT T94T and T94A variant L-FABP and cultured primary human hepatocytes. Non-conse...

  13. CooVar: Co-occurring variant analyzer

    Directory of Open Access Journals (Sweden)

    Vergara Ismael A

    2012-11-01

    Full Text Available Abstract Background Evaluating the impact of genomic variations (GV on protein-coding transcripts is an important step in identifying variants of functional significance. Currently available programs for variant annotation depend on external databases or annotate multiple variants affecting the same transcript independently, which limits program use to organisms available in these databases or results in potentially incorrect or incomplete annotations. Findings We have developed CooVar (Co-occurring Variant Analyzer, a database-independent program for assessing the impact of GVs on protein-coding transcripts. CooVar takes GVs, reference genome sequence, and protein-coding exons as input and provides annotated GVs and transcripts as output. Other than similar programs, CooVar considers the combined impact of all GVs affecting the same transcript, generating biologically more accurate annotations. CooVar is operated from the command-line and supports standard file formats VCF, GFF/GTF, and GVF, which makes it easy to integrate into existing computational pipelines. We have extensively tested CooVar on worm and human data sets and demonstrate that it generates correct annotations in only a short amount of time. Conclusions CooVar is an easy-to-use and lightweight variant annotation tool that considers the combined impact of GVs on protein-coding transcripts. CooVar is freely available at http://genome.sfu.ca/projects/coovar/.

  14. Functional modulation of the glutamate transporter variant GLT1b by the PDZ domain protein PICK1

    DEFF Research Database (Denmark)

    Søgaard, Rikke; Borre, Lars; Braunstein, Thomas H

    2013-01-01

    effect on trafficking of AMPA-type glutamate receptors. The 11 extreme C-terminal residues specific for the GLT1b variant are essential for its specific interaction with the PICK1 PDZ domain, but a functional consequence of this interaction has remained unresolved. To identify a functional effect of PICK...

  15. A novel recombinant variant of latent membrane protein 1 from Epstein Barr virus in Argentina denotes phylogeographical association

    Science.gov (United States)

    Gantuz, Magdalena; Lorenzetti, Mario Alejandro; Chabay, Paola Andrea

    2017-01-01

    Epstein Barr virus (EBV) infection in Argentina occurs at an early age and occasionally develops infectious mononucleosis (IM). EBV is also related with lymphomas. LMP1, the viral oncoprotein is polymorphic and is used to define viral variants. Aim To study LMP1 variants distribution among children with EBV+ malignant and benign conditions as well as in healthy carriers. Methods Oral secretions and blood cells from 31 children with IM, and biopsies from 14 EBV+ reactive lymphoid hyperplasia and 33 EBV+ lymphomas were included. LMP1 was amplified by nested PCR and sequenced. Phylogenetic reconstructions were made under Maximun Likelihood, Bayesian and coalescent algorithms. Results Six clades were defined (China1, China2, Med-, Alaskan, B95.8 and Argentine). Argentine variants, the most prevalent (46%), harbored 3 distinctive mutations and were a recombination between Raji and China1. Despite no pathology or compartment associations were observed for LMP1, the Argentine clade showed a phylogeographic association with our region. LMP1 estimated evolution rate was 8.591x10-5s/s/y and the estimated tMRCA for Raji and Argentine was 136ybp. Conclusions An LMP1 Argentine clade was defined. LMP1 evolutionary rate was higher than expected for herpesviruses. The tMRCA for Raji and the Argentine agrees with African immigration and could explain the recombinant nature of the Argentine variant. PMID:28328987

  16. KinMutRF: a random forest classifier of sequence variants in the human protein kinase superfamily

    DEFF Research Database (Denmark)

    Pons, Tirso; Vazquez, Miguel; Matey-Hernandez, María Luisa

    2016-01-01

    as a random forest ponder a battery of features that characterize the variants: a) at the gene level, including membership to a Kinbase group and Gene Ontology terms; b) at the PFAM domain level; and c) at the residue level, the types of amino acids involved, changes in biochemical properties, functional...

  17. denovo-db: a compendium of human de novo variants.

    Science.gov (United States)

    Turner, Tychele N; Yi, Qian; Krumm, Niklas; Huddleston, John; Hoekzema, Kendra; F Stessman, Holly A; Doebley, Anna-Lisa; Bernier, Raphael A; Nickerson, Deborah A; Eichler, Evan E

    2017-01-04

    Whole-exome and whole-genome sequencing have facilitated the large-scale discovery of de novo variants in human disease. To date, most de novo discovery through next-generation sequencing focused on congenital heart disease and neurodevelopmental disorders (NDDs). Currently, de novo variants are one of the most significant risk factors for NDDs with a substantial overlap of genes involved in more than one NDD. To facilitate better usage of published data, provide standardization of annotation, and improve accessibility, we created denovo-db (http://denovo-db.gs.washington.edu), a database for human de novo variants. As of July 2016, denovo-db contained 40 different studies and 32,991 de novo variants from 23,098 trios. Database features include basic variant information (chromosome location, change, type); detailed annotation at the transcript and protein levels; severity scores; frequency; validation status; and, most importantly, the phenotype of the individual with the variant. We included a feature on our browsable website to download any query result, including a downloadable file of the full database with additional variant details. denovo-db provides necessary information for researchers to compare their data to other individuals with the same phenotype and also to controls allowing for a better understanding of the biology of de novo variants and their contribution to disease. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Common genetic variants explain the majority of the correlation between height and intelligence: the generation Scotland study.

    Science.gov (United States)

    Marioni, Riccardo E; Batty, G David; Hayward, Caroline; Kerr, Shona M; Campbell, Archie; Hocking, Lynne J; Porteous, David J; Visscher, Peter M; Deary, Ian J

    2014-03-01

    Greater height and higher intelligence test scores are predictors of better health outcomes. Here, we used molecular (single-nucleotide polymorphism) data to estimate the genetic correlation between height and general intelligence (g) in 6,815 unrelated subjects (median age 57, IQR 49-63) from the Generation Scotland: Scottish Family Health Study cohort. The phenotypic correlation between height and g was 0.16 (SE 0.01). The genetic correlation between height and g was 0.28 (SE 0.09) with a bivariate heritability estimate of 0.71. Understanding the molecular basis of the correlation between height and intelligence may help explain any shared role in determining health outcomes. This study identified a modest genetic correlation between height and intelligence with the majority of the phenotypic correlation being explained by shared genetic influences.

  19. Polymorphisms and variants in the prion protein sequence of European moose (Alces alces), reindeer (Rangifer tarandus), roe deer (Capreolus capreolus) and fallow deer (Dama dama) in Scandinavia.

    Science.gov (United States)

    Wik, Lotta; Mikko, Sofia; Klingeborn, Mikael; Stéen, Margareta; Simonsson, Magnus; Linné, Tommy

    2012-07-01

    The prion protein (PrP) sequence of European moose, reindeer, roe deer and fallow deer in Scandinavia has high homology to the PrP sequence of North American cervids. Variants in the European moose PrP sequence were found at amino acid position 109 as K or Q. The 109Q variant is unique in the PrP sequence of vertebrates. During the 1980s a wasting syndrome in Swedish moose, Moose Wasting Syndrome (MWS), was described. SNP analysis demonstrated a difference in the observed genotype proportions of the heterozygous Q/K and homozygous Q/Q variants in the MWS animals compared with the healthy animals. In MWS moose the allele frequencies for 109K and 109Q were 0.73 and 0.27, respectively, and for healthy animals 0.69 and 0.31. Both alleles were seen as heterozygotes and homozygotes. In reindeer, PrP sequence variation was demonstrated at codon 176 as D or N and codon 225 as S or Y. The PrP sequences in roe deer and fallow deer were identical with published GenBank sequences.

  20. Capillary electrophoresis analysis of different variants of the amyloidogenic protein β2 -microglobulin as a simple tool for misfolding and stability studies.

    Science.gov (United States)

    Bertoletti, Laura; Bisceglia, Federica; Colombo, Raffaella; Giorgetti, Sofia; Raimondi, Sara; Mangione, P Patrizia; De Lorenzi, Ersilia

    2015-10-01

    Free solution capillary electrophoresis with UV detection is here used to retrieve information on the conformational changes of wild-type β2 -microglobulin and a series of naturally and artificially created variants known to have different stability and amyloidogenic potential. Under nondenaturing conditions, the resolution of at least two folding conformers at equilibrium is obtained and a third species is detected for the less stable isoforms. Partial denaturation by using chaotropic agents such as acetonitrile or trifluoroethanol reveals that the separated peaks are at equilibrium, as the presence of less structured species is either enhanced or induced at the expenses of the native form. Reproducible CE data allow to obtain an interesting semiquantitative correlation between the peak areas observed and the protein stability. Thermal unfolding over the range 25-42°C is induced inside the capillary for the two pathogenic proteins (wtβ2 -microglobulin and D76N variant): the large differences observed upon small temperature variation draw attention on the robustness of analytical methods when dealing with proteins prone to misfolding and aggregation.

  1. Neuropathic pain in two-generation twins carrying the sodium channel Nav1.7 functional variant R1150W.

    Science.gov (United States)

    Harrer, Judith U; Uçeyler, Nurcan; Doppler, Kathrin; Fischer, Tanya Z; Dib-Hajj, Sulayman D; Waxman, Stephen G; Sommer, Claudia

    2014-10-01

    We present clinical, neuropathological, and molecular genetic findings of a family with a new pain phenotype of the sodium channel gene SCN9A polymorphism R1150W. A 46-year-old woman presented with a 5-year history of episodic temperature- and exercise-dependent burning pain of the feet and lower legs associated with numbness of the distal upper and lower limbs. Her monozygotic twin sister and their mother and her twin presented similar symptoms. Clinical evaluation was normal except for a mild distal sensory deficit in fingers and feet. Electrophysiological testing was unremarkable, as were serum and cerebrospinal fluid laboratory findings. Skin biopsies of the distal lower limbs revealed an epidermal nerve fiber density at the lower limit of normal. Myelinated dermal nerve fibers showed elongated nodes of Ranvier, but normal distribution of nodal and paranodal proteins. Genetic testing for ion channel-associated pain disorders revealed an amino acid R1150W substitution of the Nav1.7 sodium channel. The combination of a Nav1.7 polymorphism with dysmyelinating features in small-caliber peripheral nerves has not been described before and may suggest an explanation for the clinical syndrome in our patients. Treatment with the sodium channel blocker lamotrigine provided some relief, consistent with a role of sodium channel dysfunction in the pain syndrome of this family.

  2. Retention behaviour of proteins on poly(vinylimidazole)-copper(II) complexes supported on silica: application to the fractionation of desialylated human alpha 1-acid glycoprotein variants.

    Science.gov (United States)

    Millot, M C; Hervé, F; Sébille, B

    1995-02-03

    The retention behaviour of various amino acids, peptides and proteins on poly(vinylimidazole)-Cu(II) complexes supported on silica was investigated. Free amino acids and peptides containing one histidine and in some instances one additional tryptophan residue in their primary structure were found to elute from the supports only after addition of a competing complexing agent to the mobile phase. However, the results obtained the proteins containing metal binding groups suggested that, in addition to the presence of donor-acceptor interactions between the macromolecules and the immobilized metal, other additional (essentially ionic and/or hydrophobic) interactions took place between the proteins and the surrounding of the metal. When donor-acceptor interactions were predominant, proteins were strongly adsorbed on the stationary phase and their elution required the addition of a competing complexing agent in the mobile phase. However, when the binding between the proteins and the supports via donor-acceptor interactions was less favourable, proteins were eluted from the columns without the addition of a competing agent in the mobile phase. With respect to the binding of these proteins, ionic and/or hydrophobic interactions were no longer negligible during the chromatographic process and the retention of the macromolecules by the stationary phase depended on the elution conditions (ionic strength, pH, etc.). These supports were used in the fractionation of the three main genetic variants of desialylated alpha 1-acid glycoprotein.

  3. Assessment of the Fusion Tags on Increasing Soluble Production of the Active TEV Protease Variant and Other Target Proteins in E. coli.

    Science.gov (United States)

    Yu, Xuelian; Sun, Jiaqi; Wang, Weiyu; Jiang, Li; Cheng, Beijiu; Fan, Jun

    2016-12-17

    In this study, five fusion tags affecting soluble production and cleavage activity of the tobacco etch virus (TEV) protease (TEVp) variant in Escherichia coli strains BL21 (DE3) and Rosetta™ (DE3) are investigated. Combination of the augmenting rare transfer RNAs (tRNAs) and the fused expressivity tag (N-terminal seven amino acid residues of E. coli translation initiation factor II) promotes the soluble TEVp partner expressed at relatively high level. Attachment of the maltose-binding protein (MBP) tag increases soluble expression of the protease released from the fusion protein in E. coli cells, but the incorporated TEVp recognition sequence slightly decreases expressivity of the fusion construct. Except for the green fluorescent protein, the attached expressivity tag shows less efficiency than the MBP tag in enhancing expression levels of the selected five target proteins in the Rosetta™ (DE3) cells under different induction conditions. Our results identified that high-level production of the functional target protein as the fusion partner in E. coli is combined with the intrinsic property of fusion tag, fusion protein stability, inherent folding of target protein, rare tRNA abundance, and the incorporated linker. Purified TEVp fusion constructs with the N-terminal expressivity tag, as well as the MBP partner, are the ideal alternatives for removing fusion tag.

  4. Generation of transgenic dogs that conditionally express green fluorescent protein.

    Science.gov (United States)

    Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Hong, So Gun; Jang, Goo; Kwon, Mo Sun; Koo, Bon Chul; Kim, Teoan; Kang, Sung Keun; Ra, Jeong Chan; Ko, Chemyong; Lee, Byeong Chun

    2011-06-01

    We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies.

  5. Characterization of mutations and sequence variants in the D21S11 locus by next generation sequencing

    DEFF Research Database (Denmark)

    Rockenbauer, Eszter; Hansen, Stine; Mikkelsen, Martin;

    2014-01-01

    We sequenced the D21S11 locus in 77 individuals from Danish paternity cases using 454 FLX next generation sequencing (NGS) technology. All samples were also typed with the AmpFlSTR(®) Profiler Plus(®) or the AmpFlSTR(®) Identifiler(®) PCR Amplification kits as part of paternity investigations....... In 18 of the confirmed trios, a genetic inconsistency was observed between one of the parents and the child at the D21S11 locus. NGS of the D21S11 locus revealed which allele had mutated from which parent to the child in 13 of these trios. All characterized mutations could be explained by single......-step mutations in the longest sub-repeat of D21S11. A total of 53 of the 77 sequenced samples originated from unrelated individuals. Twenty different D21S11 alleles were detected by NGS in these individuals whereas only 13 different alleles were observed with fragment analysis. Several alleles had the same...

  6. BRCA1-2 diagnostic workflow from next-generation sequencing technologies to variant identification and final report.

    Science.gov (United States)

    Pilato, Brunella; Pinto, Rosamaria; De Summa, Simona; Petriella, Daniela; Lacalamita, Rosanna; Danza, Katia; Paradiso, Angelo; Tommasi, Stefania

    2016-10-01

    The BRCA1-BRCA2 genes predispose to hereditary breast and ovarian cancer, and the germline and mutational status of these genes defines a target population that can benefit from PARP inhibitor treatments. To respond to the increasing number of BRCA1-BRCA2 tests, it is necessary to shift to high-throughput technologies that are reliable and less time consuming. Different methodological platforms are dedicated to this purpose with different approaches and algorithms for analysis. Our aim was to set up a cost-effective and low time-consuming BRCA1-BRCA2 mutation detection workflow using the Ion Torrent PGM technology. A retrospective cohort of 40 patients with familial breast/ovarian cancer previously tested by Sanger sequencing and a prospective cohort of 72 patients (validation set) were analyzed. The validation set included 64 patients affected by familial breast/ovarian cancer and eight sporadic ovarian cancer cases, who are potential candidates for PARPi treatments. A complete and standardized workflow easily usable and suitable in a certified laboratory has been proved and validated. This includes all steps from library preparation to the final report. The use of next-generation sequencing will be of benefit for patients enrolled in the genetic counseling process and, moreover, will enhance the process of selecting patients eligible for personalized treatments. © 2016 Wiley Periodicals, Inc.

  7. Identifying actionable variants using next generation sequencing in patients with a historical diagnosis of undifferentiated pleomorphic sarcoma.

    Science.gov (United States)

    Lewin, Jeremy; Garg, Swati; Lau, Beatrice Y; Dickson, Brendan C; Traub, Frank; Gokgoz, Nalan; Griffin, Anthony M; Ferguson, Peter C; Andrulis, Irene L; Sim, Hao-Wen; Kamel-Reid, Suzanne; Stockley, Tracy L; Siu, Lillian L; Wunder, Jay S; Razak, Albiruni R A

    2017-09-10

    There are limited data regarding the molecular characterization of undifferentiated pleomorphic sarcomas (UPS; formerly malignant fibrous histiocytoma). This study aimed to investigate the utility of next generation sequencing (NGS) in UPS to identify subsets of patients who harbour actionable mutations. Patients diagnosed with UPS underwent pathological re-evaluation by a pathologist specializing in sarcoma. Tumor DNA was isolated from archived fresh frozen tissue samples and genotyped using NGS with the Illumina MiSeq TruSeq Amplicon Cancer Panel (48 genes, 212 amplicons). In total, 95 patients initially classified with UPS were identified. Following pathology re-review the histological subtypes were reclassified to include: Myxofibrosarcoma (MFS, N = 44); UPS(N = 18); and Others (N = 27; including undifferentiated spindle cell sarcoma (N = 15) and dedifferentiated liposarcoma (N = 6)). Seven cases were excluded from further analysis for other reasons. Baseline demographics of the finalized cohort (N = 88) showed a median age of 66 years (32-95), primarily with stage I-III disease (92%) and high-grade (86%) lesions. Somatic mutations were identified in 31 cases (35%)(Total mutations = 36: solitary mutation(n = 27); two mutations( =n = 3); three mutations(n = 1)). The most commonly identified mutations were in TP53 (n = 24), ATM (n = 3) and PIK3CA (n = 2). Three of 43 patients with MFS and one of 18 patients with UPS had clinically relevant mutations, mainly related to biomarkers of prediction of response; however few had targetable driver mutations. Somatic mutation status did not influence disease free or overall survival. Based on the small number of clinically relevant mutations, these data do not support the routine use of targeted NGS panels outside of research protocols in UPS. © 2017 UICC.

  8. Generation of the regulatory protein rtTA transgenic mice

    Institute of Scientific and Technical Information of China (English)

    Kang Xu; Xin-Yan Deng; Ying Yue; Zhong-Min Guo; Bing Huang; Xun Hong; Dong Xiao; Xi-Gu Chen

    2005-01-01

    AIM: To translate Tet-on system into a conditional mouse model, in which hepatitis B or C virus (HBV or HCV) gene could be spatiotemporally expressed to overcome "immune tolerance" formed during the embryonic development and "immune escape" against hepatitis virus antigen(s), an effector mouse, carrying the reverse tetracycline-responsive transcriptional activator (rtTA) gene under the tight control of liver-specific human apoE promoter, is required to be generated. METHODS: To address this end, rtTA fragment amplified by PCR was effectively inserted into the vector of pLiv.7 containing apoE promoter to create the rtTA expressing vector, I.e., pApoE-rtTA. ApoE-rtTA transgenic fragment (-6.9 kb) released from pApoE-rtTA was transferred into mice by pronucleus injection, followed by obtaining one transgene (+) founder animal from microinjection through PCR and Southern blot analysis.RESULTS: rtTA transgene which could be transmitted to subsequent generation (F1) derived from founder was expressed in a liver-specific fashion. CONCLUSION: Taken together, these findings demonstrate that rtTA transgenic mice, in which rtTA expression is appropriately targeted to the murine liver, are successfully produced, which lays a solid foundation to 'off-on-off' regulate expression of target gene (s) (e.g., HBV and/or HCV) in transgenic mice mediated by Tet-on system.

  9. Betaine suppressed Aβ generation by altering amyloid precursor protein processing.

    Science.gov (United States)

    Liu, Xiu-Ping; Qian, Xiang; Xie, Yue; Qi, Yan; Peng, Min-Feng; Zhan, Bi-Cui; Lou, Zheng-Qing

    2014-07-01

    Betaine was an endogenous catabolite of choline, which could be isolated from vegetables and marine products. Betaine could promote the metabolism of homocysteine in healthy subjects and was used for hyperlipidemia, coronary atherosclerosis, and fatty liver in clinic. Recent findings shown that Betaine rescued neuronal damage due to homocysteine induced Alzheimer's disease (AD) like pathological cascade, including tau hyperphosphorylation and amyloid-β (Aβ) deposition. Aβ was derived from amyloid precursor protein (APP) processing, and was a triggering factor for AD pathological onset. Here, we demonstrated that Betaine reduced Aβ levels by altering APP processing in N2a cells stably expressing Swedish mutant of APP. Betaine increased α-secretase activity, but decreased β-secretase activity. Our data indicate that Betaine might play a protective role in Aβ production.

  10. Unveiling how an archetypal fluorescent protein operates: theoretical perspective on the ultrafast excited state dynamics of GFP variant S65T/H148D.

    Science.gov (United States)

    Armengol, Pau; Gelabert, Ricard; Moreno, Miquel; Lluch, José M

    2015-02-12

    Green fluorescent protein variant S65T/H148D has been reported to host a photocycle involving the photoinduced proton transfer reaction between the chromophore and residue Asp148 under 50 fs and without a measurable kinetic isotope effect, and experimental evidence is suggestive of the existence of a highly delocalized proton between these residues. The blinding speed at which this biological system undergoes proton transfer has been ascribed to the extreme increase of acidity of the GFP chromophore in the electronic excited state where proton transfer takes place. This work strives to present a coherent, complete, and balanced description of the dynamics of this specific variant of GFP in which it will be shown that this increase of acidity is insufficient to explain the behavior observed. This study tracks the behavior of this photosystem to the delicate interplay between structure and dynamics shown in the presence of solvent. In this way, it has been found that the dynamics of this protein intertwines its structure with the intervening solvent to give rise to effectively degenerate situations in what concerns the reactants and products of the proton transfer reaction in ground and, most importantly, photoexcited state, in terms of potential energy profiles associated with the proton migration. Under these conditions, proton transfer can occur in accordance with the experimental data available. This set of characteristics is possibly common to a host of other proton transfer based fluorescent proteins, and helps promoting GFP S65T/H148D to a case of archetypal significance. Thus, our results can be useful to understand the way many fluorescent proteins work and, more generally, the molecular basis for proton transfer reactions in proteins.

  11. Sent packing: protein engineering generates a new crystal form of Pseudomonas aeruginosa DsbA1 with increased catalytic surface accessibility

    Energy Technology Data Exchange (ETDEWEB)

    McMahon, Roisin M., E-mail: r.mcmahon1@uq.edu.au; Coinçon, Mathieu; Tay, Stephanie; Heras, Begoña [University of Queensland, 306 Carmody Road, Brisbane, Queensland 4072 (Australia); Morton, Craig J. [Biota Holdings Limited, Unit 10, 585 Blackburn Road, Notting Hill, Victoria 3168 (Australia); Scanlon, Martin J. [Monash University, 381 Royal Parade, Parkville, Victoria 3052 (Australia); Martin, Jennifer L. [University of Queensland, 306 Carmody Road, Brisbane, Queensland 4072 (Australia)

    2015-11-26

    The crystal structure of a P. aeruginosa DsbA1 variant is more suitable for fragment-based lead discovery efforts to identify inhibitors of this antimicrobial drug target. In the reported structures the active site of the protein can simultaneously bind multiple ligands introduced in the crystallization solution or via soaking. Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discovery approach to PaDsbA1 inhibitor development, the identification of a crystal form of PaDsbA1 that was more suitable for fragment-soaking experiments was sought. A previously identified crystallization condition for this protein was unsuitable, as in this crystal form of PaDsbA1 the active-site surface loops are engaged in the crystal packing, occluding access to the target site. A single residue involved in crystal-packing interactions was substituted with an amino acid commonly found at this position in closely related enzymes, and this variant was successfully used to generate a new crystal form of PaDsbA1 in which the active-site surface is more accessible for soaking experiments. The PaDsbA1 variant displays identical redox character and in vitro activity to wild-type PaDsbA1 and is structurally highly similar. Two crystal structures of the PaDsbA1 variant were determined in complex with small molecules bound to the protein active site. These small molecules (MES, glycerol and ethylene glycol) were derived from the crystallization or cryoprotectant solutions and provide a proof of principle that the reported crystal form will be amenable to co-crystallization and soaking with small molecules designed to target the protein active-site surface.

  12. A second-generation protein-protein interaction network of Helicobacter pylori.

    Science.gov (United States)

    Häuser, Roman; Ceol, Arnaud; Rajagopala, Seesandra V; Mosca, Roberto; Siszler, Gabriella; Wermke, Nadja; Sikorski, Patricia; Schwarz, Frank; Schick, Matthias; Wuchty, Stefan; Aloy, Patrick; Uetz, Peter

    2014-05-01

    Helicobacter pylori infections cause gastric ulcers and play a major role in the development of gastric cancer. In 2001, the first protein interactome was published for this species, revealing over 1500 binary protein interactions resulting from 261 yeast two-hybrid screens. Here we roughly double the number of previously published interactions using an ORFeome-based, proteome-wide yeast two-hybrid screening strategy. We identified a total of 1515 protein-protein interactions, of which 1461 are new. The integration of all the interactions reported in H. pylori results in 3004 unique interactions that connect about 70% of its proteome. Excluding interactions of promiscuous proteins we derived from our new data a core network consisting of 908 interactions. We compared our data set to several other bacterial interactomes and experimentally benchmarked the conservation of interactions using 365 protein pairs (interologs) of E. coli of which one third turned out to be conserved in both species.

  13. Allelic diversity of the Plasmodium falciparum erythrocyte membrane protein 1 entails variant-specific red cell surface epitopes.

    Directory of Open Access Journals (Sweden)

    Inès Vigan-Womas

    Full Text Available The clonally variant Plasmodium falciparum PfEMP1 adhesin is a virulence factor and a prime target of humoral immunity. It is encoded by a repertoire of functionally differentiated var genes, which display architectural diversity and allelic polymorphism. Their serological relationship is key to understanding the evolutionary constraints on this gene family and rational vaccine design. Here, we investigated the Palo Alto/VarO and IT4/R29 and 3D7/PF13_003 parasites lines. VarO and R29 form rosettes with uninfected erythrocytes, a phenotype associated with severe malaria. They express an allelic Cys2/group A NTS-DBL1α(1 PfEMP1 domain implicated in rosetting, whose 3D7 ortholog is encoded by PF13_0003. Using these three recombinant NTS-DBL1α(1 domains, we elicited antibodies in mice that were used to develop monovariant cultures by panning selection. The 3D7/PF13_0003 parasites formed rosettes, revealing a correlation between sequence identity and virulence phenotype. The antibodies cross-reacted with the allelic domains in ELISA but only minimally with the Cys4/group B/C PFL1955w NTS-DBL1α. By contrast, they were variant-specific in surface seroreactivity of the monovariant-infected red cells by FACS analysis and in rosette-disruption assays. Thus, while ELISA can differentiate serogroups, surface reactivity assays define the more restrictive serotypes. Irrespective of cumulated exposure to infection, antibodies acquired by humans living in a malaria-endemic area also displayed a variant-specific surface reactivity. Although seroprevalence exceeded 90% for each rosetting line, the kinetics of acquisition of surface-reactive antibodies differed in the younger age groups. These data indicate that humans acquire an antibody repertoire to non-overlapping serotypes within a serogroup, consistent with an antibody-driven diversification pressure at the population level. In addition, the data provide important information for vaccine design, as

  14. No evidence that protein truncating variants in BRIP1 are associated with breast cancer risk: Implications for gene panel testing

    NARCIS (Netherlands)

    D.F. Easton (Douglas F.); F. Lesueur (Fabienne); B. Decker (Brennan); K. Michailidou (Kyriaki); J. Li (Jun); J. Allen (Jamie); C. Luccarini (Craig); K.A. Pooley (Karen); M. Shah (Mitul); M.K. Bolla (Manjeet); Q. Wang (Qin); J. Dennis (Joe); J. Ahmad (Jamil); E.R. Thompson (Ella); F. Damiola (Francesca); M. Pertesi (Maroulio); C. Voegele (Catherine); N. Mebirouk (Noura); N. Robinot (Nivonirina); G. Durand (Geoffroy); N. Forey (Nathalie); R.N. Luben (Robert); S. Ahmed (Shahana); K. Aittomäki (Kristiina); H. Anton-Culver (Hoda); V. Arndt (Volker); C. Baynes (Caroline); M.W. Beckman (Matthias W.); J. Benítez (Javier); D. Van Den Berg (David); W.J. Blot (William); N.V. Bogdanova (Natalia); S.E. Bojesen (Stig); H. Brenner (Hermann); J. Chang-Claude (Jenny); K.S. Chia (Kee Seng); J.-Y. Choi (Ji-Yeob); D. Conroy (Don); A. Cox (Angela); S.S. Cross (Simon S.); K. Czene (Kamila); H. Darabi (Hatef); P. Devilee (Peter); M. Eriksson (Mats); P.A. Fasching (Peter); J.D. Figueroa (Jonine); H. Flyger (Henrik); F. Fostira (Florentia); M. García-Closas (Montserrat); G.G. Giles (Graham); G. Glendon (Gord); A. González-Neira (Anna); P. Guénel (Pascal); C.A. Haiman (Christopher A.); P. Hall (Per); S.N. Hart (Steven N.); J.M. Hartman (Joost); M.J. Hooning (Maartje); C.-N. Hsiung (Chia-Ni); H. Ito (Hidemi); A. Jakubowska (Anna); P.A. James (Paul A.); E.M. John (Esther M.); N. Johnson (Nichola); M. Jones (Michael); M. Kabisch (Maria); D. Kang (Daehee); V-M. Kosma (Veli-Matti); V. Kristensen (Vessela); D. Lambrechts (Diether); N. Li (Na); E. Myöhänen (Eija); H. Kemiläinen (Helena); A. Lindblom (Annika); J. Long (Jirong); A. Lophatananon (Artitaya); J. Lubinski (Jan); A. Mannermaa (Arto); K. Matsuo (Keitaro); S. Margolin (Sara); K. Matsuo (Keitaro); A. Meindl (Alfons); G. Mitchell (Gillian); K.R. Muir (K.); I. Nevelsteen (Ines); A.M.W. van den Ouweland (Ans); P. Peterlongo (Paolo); S.-Y. Phuah (Sze-Yee); K. Pykäs (Katri); S.M. Rowley (Simone M.); C-Y. Shen (Chen-Yang); R.K. Schmutzler (Rita); C.-Y. Shen (Chen-Yang); X.-O. Shu (Xiao-Ou); M.C. Southey (Melissa); H. Surowy (Harald); A.J. Swerdlow (Anthony ); S.-H. Teo; R.A.E.M. Tollenaar (Rob); I.P. Tomlinson (Ian); C. Vachon (Celine); T. Truong (Thérèse); C. Vachon (Celine); S. Verhoef; M. Wong-Brown (Michelle); W. Zheng (Wei); Y. Zheng (Ying); H. Nevanlinna (Heli); R.J. Scott (Rodney); I.L. Andrulis (Irene); F.J. Couch (Fergus); J.L. Hopper (John); B. Burwinkel (Barbara); R. Winqvist (Robert); B. Burwinkel (Barbara); E.J. Sawyer (Elinor); M.K. Schmidt (Marjanka); A. Rudolph (Anja); T. Dörk (Thilo); S.L. Neuhausen (Susan); U. Hamann (Ute); S.L. Neuhausen (Susan L.); R.L. Milne (Roger); O. Fletcher (Olivia); P.D.P. Pharoah (Paul); I. Campbell (Ian); D. Goldgar (David); S.V. Tavtigian (Sean); D.E. Goldgar (David E.); S.V. Tavtigian (Sean V.); G. Chenevix-Trench (Georgia); K. Kleivi (Kristine); L. Ottestad (Lars); R. Karesen (Rolf); A. Langerød (Anita); E. Schlichting (Ellen); M.M. Holmen (Marit Muri); T. Sauer (Toril); V. Haakensen (Vilde); O. Engebråten (Olav); B. Naume (Bjorn); C.E. Kiserud (Cecile E.); K.V. Reinertsen (Kristin V.); Å. Helland (Åslaug); M. Riis (Margit); I. Bukholm (Ida); P.E. Lønning (Per ); S. Nord (Silje); G.G. Alnæs (Grethe Grenaker)

    2016-01-01

    textabstractBackground BRCA1 interacting protein C-terminal helicase 1 (BRIP1) is one of the Fanconi Anaemia Complementation (FANC) group family of DNA repair proteins. Biallelic mutations in BRIP1 are responsible for FANC group J, and previous studies have also suggested that rare protein

  15. Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases

    DEFF Research Database (Denmark)

    Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

    2008-01-01

    The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were...

  16. HistoneDB 2.0: a histone database with variants--an integrated resource to explore histones and their variants.

    Science.gov (United States)

    Draizen, Eli J; Shaytan, Alexey K; Mariño-Ramírez, Leonardo; Talbert, Paul B; Landsman, David; Panchenko, Anna R

    2016-01-01

    Compaction of DNA into chromatin is a characteristic feature of eukaryotic organisms. The core (H2A, H2B, H3, H4) and linker (H1) histone proteins are responsible for this compaction through the formation of nucleosomes and higher order chromatin aggregates. Moreover, histones are intricately involved in chromatin functioning and provide a means for genome dynamic regulation through specific histone variants and histone post-translational modifications. 'HistoneDB 2.0--with variants' is a comprehensive database of histone protein sequences, classified by histone types and variants. All entries in the database are supplemented by rich sequence and structural annotations with many interactive tools to explore and compare sequences of different variants from various organisms. The core of the database is a manually curated set of histone sequences grouped into 30 different variant subsets with variant-specific annotations. The curated set is supplemented by an automatically extracted set of histone sequences from the non-redundant protein database using algorithms trained on the curated set. The interactive web site supports various searching strategies in both datasets: browsing of phylogenetic trees; on-demand generation of multiple sequence alignments with feature annotations; classification of histone-like sequences and browsing of the taxonomic diversity for every histone variant. HistoneDB 2.0 is a resource for the interactive comparative analysis of histone protein sequences and their implications for chromatin function. Database URL: http://www.ncbi.nlm.nih.gov/projects/HistoneDB2.0.

  17. Blocking protein farnesylation improves nuclear shape abnormalities in keratinocytes of mice expressing the prelamin A variant in Hutchinson-Gilford progeria syndrome.

    Science.gov (United States)

    Wang, Yuexia; Ostlund, Cecilia; Worman, Howard J

    2010-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by mutations in LMNA leading to expression of a truncated prelamin A variant termed progerin. Whereas a farnesylated polypeptide is normally removed from the carboxyl-terminus of prelamin A during endoproteolytic processing to lamin A, progerin lacks the cleavage site and remains farnesylated. Cultured cells from human subjects with HGPS and genetically modified mice expressing progerin have nuclear morphological abnormalities, which are reversed by inhibitors of protein farnesylation. In addition, treatment with protein farnesyltransferase inhibitors improves whole animal phenotypes in mouse models of HGPS. However, improvement in nuclear morphology in tissues after treatment of animals has not been demonstrated. We therefore treated transgenic mice that express progerin in epidermis with the protein farnesyltransferase inhibitor FTI-276 or a combination of pravastatin and zoledronate to determine if they reversed nuclear morphological abnormalities in tissue. Immunofluorescence microscopy and "blinded" electron microscopic analysis demonstrated that systemic administration of FTI-276 or pravastatin plus zoledronate significantly improved nuclear morphological abnormalities in keratinocytes of transgenic mice. These results show that pharmacological blockade of protein prenylation reverses nuclear morphological abnormalities that occur in HGPS in vivo. They further suggest that skin biopsy may be useful to determine if protein farnesylation inhibitors are exerting effects in subjects with HGPS in clinical trials.

  18. Generation of induced pluripotent stem cells (iPSCs from a hypertrophic cardiomyopathy patient with the pathogenic variant p.Val698Ala in beta-myosin heavy chain (MYH7 gene

    Directory of Open Access Journals (Sweden)

    Samantha Barratt Ross

    2017-04-01

    Full Text Available Induced pluripotent stem cells (iPSCs were generated from peripheral blood mononuclear cells (PBMCs isolated from the whole blood of a 43-year-old male with hypertrophic cardiomyopathy (HCM who carries the pathogenic variant p.Val698Ala in beta-myosin heavy chain (MYH7. Patient-derived PBMCs were reprogrammed using non-integrative episomal vectors containing reprogramming factors OCT4, SOX2, LIN28, KLF4 and L-MYC. iPSCs were shown to express pluripotent markers, have trilineage differentiation potential, carry the pathogenic MYH7 variant p.Val698Ala, have a normal karyotype and no longer carry the episomal reprogramming vector. This line is useful for studying the link between variants in MYH7 and the pathogenesis of HCM.

  19. G-protein beta3 subunit gene variant is unlikely to have a significant influence on serum uric acid level in Japanese workers.

    Science.gov (United States)

    Suwazono, Yasushi; Kobayashi, Etsuko; Uetani, Mirei; Miura, Katsuyuki; Morikawa, Yuko; Ishizaki, Masao; Kido, Teruhiko; Nakagawa, Hideaki; Nogawa, Koji

    2006-06-01

    The C825T variant of the G-protein beta3 subunit (GNB3) gene has attracted renewed attention as a candidate gene for obesity, hypertension and hyperuricemia. The main role of G-protein is to translate signals from the cell surface into a cellular response. The 825T allele is associated with a splice variant of GNB3 protein and enhanced G-protein activation. We examined the relationship between this variant and the risk of hyperuricemia in Japanese workers. The study subjects were 1,452 men and 1,169 women selected from 3,834 men and 2,591 women in 1997. On the basis of common clinical criteria, hyperuricemia I was defined as serum uric acid >or= 7.0 mg/dl in men and 6.0 mg/dl in women or taking antihyperuricemic medication. The hyperuricemia I group consisted of 186 men and 20 women and its control of 1,266 men and 1,149 women. Hyperuricemia II was defined as serum uric acid > 5.7 mg/dl (median) in men and 3.9 mg/dl (median) in women or taking antihyperuricemic medication. The hyperuricemic II group consisted of 684 men and 570 women and its control of 768 men and 599 women. To replicate previous significant results in young Caucasian men, we selected these criteria because the authors of the study in young Caucasian men adopted the median in their subjects as a cut-off. The statistical power was estimated as 99% based on the significant results in Caucasians. Genotype and allele distributions in men and women with hyperuricemia I and II were not significantly different from those in the corresponding control groups. Logistic regression analysis on hyperuricemia I and II, and multiple regression on serum uric acid level demonstrated no significant effect of the C825T genotype. Despite the sufficient statistical power, this study could not demonstrate the significant influence of C825T on hyperuricemia or serum uric acid. The targeting of this polymorphism is unlikely to be beneficial in the prevention of hyperuricemia in the general Japanese population.

  20. Homology Modeling: Generating Structural Models to Understand Protein Function and Mechanism

    Science.gov (United States)

    Ramachandran, Srinivas; Dokholyan, Nikolay V.

    Geneticists and molecular and cell biologists routinely uncover new proteins important in specific biological processes/pathways. However, either the molecular functions or the functional mechanisms of many of these proteins are unclear due to a lack of knowledge of their atomic structures. Yet, determining experimental structures of many proteins presents technical challenges. The current methods for obtaining atomic-resolution structures of biomolecules (X-ray crystallography and NMR spectroscopy) require pure preparations of proteins at concentrations much higher than those at which the proteins exist in a physiological environment. Additionally, NMR has size limitations, with current technology limited to the determination of structures of proteins with masses of up to 15 kDa. Due to these reasons, atomic structures of many medically and biologically important proteins do not exist. However, the structures of these proteins are essential for several purposes, including in silico drug design [1], understanding the effects of disease mutations [2], and designing experiments to probe the functional mechanisms of proteins. Comparative modeling has gained importance as a tool for bridging the gap between sequence and structure space, allowing researchers to build structural models of proteins that are difficult to crystallize or for which structure determination by NMR spectroscopy is not tractable. Comparative modeling, or homology modeling, exploits the fact that two proteins whose sequences are evolutionarily connected display similar structural features [3]. Thus, the known structure of a protein (template) can be used to generate a molecular model of the protein (query) whose experimental structure is notknown.

  1. The Alzheimer's Disease-Associated R47H Variant of TREM2 Has an Altered Glycosylation Pattern and Protein Stability

    Science.gov (United States)

    Park, Ji-Seon; Ji, In Jung; Kim, Dong-Hou; An, Hyun Joo; Yoon, Seung-Yong

    2017-01-01

    The R47H coding variant of the triggering receptor expressed on myeloid cells-2 (TREM2) increases the risk of Alzheimer's disease (AD) similar to apolipoprotein E4. TREM2 R47H has recently been shown to have impaired binding to damage-associated lipid or apolipoprotein ligands. However, it is not known how this R47H variant affects the biochemical characteristics of TREM2 and alters the pathogenesis of AD. We previously reported that TREM2-R47H has a slightly different glycosylation pattern from wild-type. A more detailed characterization in our present study confirms that TREM2 R47H has an altered glycosylation pattern and reduced stability. TREM2 R47H shows different glycosylation profiles from analysis using monensin or kifunensine treatment which were confirmed by mass spectrometry. The solubility of TREM2 R47H and its cleaved products such as intracellular domain (ICD) is also decreased, increasing its proteasomal and lysosomal degradation. The different biochemical characteristics of TREM2 R47H, including glycosylation, solubility and processing, may offer insights into a future therapeutic strategy for AD. PMID:28149270

  2. Interpreting functional effects of coding variants: challenges in proteome-scale prediction, annotation and assessment.

    Science.gov (United States)

    Shameer, Khader; Tripathi, Lokesh P; Kalari, Krishna R; Dudley, Joel T; Sowdhamini, Ramanathan

    2016-09-01

    Accurate assessment of genetic variation in human DNA sequencing studies remains a nontrivial challenge in clinical genomics and genome informatics. Ascribing functional roles and/or clinical significances to single nucleotide variants identified from a next-generation sequencing study is an important step in genome interpretation. Experimental characterization of all the observed functional variants is yet impractical; thus, the prediction of functional and/or regulatory impacts of the various mutations using in silico approaches is an important step toward the identification of functionally significant or clinically actionable variants. The relationships between genotypes and the expressed phenotypes are multilayered and biologically complex; such relationships present numerous challenges and at the same time offer various opportunities for the design of in silico variant assessment strategies. Over the past decade, many bioinformatics algorithms have been developed to predict functional consequences of single nucleotide variants in the protein coding regions. In this review, we provide an overview of the bioinformatics resources for the prediction, annotation and visualization of coding single nucleotide variants. We discuss the currently available approaches and major challenges from the perspective of protein sequence, structure, function and interactions that require consideration when interpreting the impact of putatively functional variants. We also discuss the relevance of incorporating integrated workflows for predicting the biomedical impact of the functionally important variations encoded in a genome, exome or transcriptome. Finally, we propose a framework to classify variant assessment approaches and strategies for incorporation of variant assessment within electronic health records.

  3. Protein engineering for metabolic engineering: current and next-generation tools

    Science.gov (United States)

    Marcheschi, Ryan J.; Gronenberg, Luisa S.; Liao, James C.

    2014-01-01

    Protein engineering in the context of metabolic engineering is increasingly important to the field of industrial biotechnology. As the demand for biologically-produced food, fuels, chemicals, food additives, and pharmaceuticals continues to grow, the ability to design and modify proteins to accomplish new functions will be required to meet the high productivity demands for the metabolism of engineered organisms. This article reviews advances of selecting, modeling, and engineering proteins to improve or alter their activity. Some of the methods have only recently been developed for general use and are just beginning to find greater application in the metabolic engineering community. We also discuss methods of generating random and targeted diversity in proteins to generate mutant libraries for analysis. Recent uses of these techniques to alter cofactor use, produce non-natural amino acids, alcohols, and carboxylic acids, and alter organism phenotypes are presented and discussed as examples of the successful engineering of proteins for metabolic engineering purposes. PMID:23589443

  4. Protein engineering for metabolic engineering: current and next-generation tools.

    Science.gov (United States)

    Marcheschi, Ryan J; Gronenberg, Luisa S; Liao, James C

    2013-05-01

    Protein engineering in the context of metabolic engineering is increasingly important to the field of industrial biotechnology. As the demand for biologically produced food, fuels, chemicals, food additives, and pharmaceuticals continues to grow, the ability to design and modify proteins to accomplish new functions will be required to meet the high productivity demands for the metabolism of engineered organisms. We review advances in selecting, modeling, and engineering proteins to improve or alter their activity. Some of the methods have only recently been developed for general use and are just beginning to find greater application in the metabolic engineering community. We also discuss methods of generating random and targeted diversity in proteins to generate mutant libraries for analysis. Recent uses of these techniques to alter cofactor use; produce non-natural amino acids, alcohols, and carboxylic acids; and alter organism phenotypes are presented and discussed as examples of the successful engineering of proteins for metabolic engineering purposes.

  5. Broad-Bandwidth Chiral Sum Frequency Generation Spectroscopy for Probing the Kinetics of Proteins at Interfaces.

    Science.gov (United States)

    Wang, Zhuguang; Fu, Li; Ma, Gang; Yan, Elsa C Y

    2015-10-27

    The kinetics of proteins at interfaces plays an important role in biological functions and inspires solutions to fundamental problems in biomedical sciences and engineering. Nonetheless, due to the lack of surface-specific and structural-sensitive biophysical techniques, it still remains challenging to probe protein kinetics in situ and in real time without the use of spectroscopic labels at interfaces. Broad-bandwidth chiral sum frequency generation (SFG) spectroscopy has been recently developed for protein kinetic studies at interfaces by tracking the chiral vibrational signals of proteins. In this article, we review our recent progress in kinetic studies of proteins at interfaces using broad-bandwidth chiral SFG spectroscopy. We illustrate the use of chiral SFG signals of protein side chains in the C-H stretch region to monitor self-assembly processes of proteins at interfaces. We also present the use of chiral SFG signals from the protein backbone in the N-H stretch region to probe the real-time kinetics of proton exchange between protein and water at interfaces. In addition, we demonstrate the applications of spectral features of chiral SFG that are typical of protein secondary structures in both the amide I and the N-H stretch regions for monitoring the kinetics of aggregation of amyloid proteins at membrane surfaces. These studies exhibit the power of broad-bandwidth chiral SFG to study protein kinetics at interfaces and the promise of this technique in research areas of surface science to address fundamental problems in biomedical and material sciences.

  6. A two-step protein quality control pathway for a misfolded DJ-1 variant in fission yeast

    DEFF Research Database (Denmark)

    Mathiassen, Søs Grønbæk; Larsen, Ida B.; Poulsen, Esben Guldahl

    2015-01-01

    A mutation, L166P, in the cytosolic protein, PARK7/DJ-1, causes protein misfolding and is linked to Parkinson disease. Here, we identify the fission yeast protein Sdj1 as the orthologue of DJ-1 and calculate by in silico saturation mutagenesis the effects of point mutants on its structural...... stability. We also map the degradation pathways for Sdj1-L169P, the fission yeast orthologue of the disease-causing DJ-1 L166P protein. Sdj1-L169P forms inclusions, which are enriched for the Hsp104 disaggregase. Hsp104 and Hsp70-type chaperones are required for efficient degradation of Sdj1-L169P...

  7. Polymorphism in the exon 4 of β-lactoglobulin variant B precursor gene and its association with milk traits and protein structure in Chinese Holstein.

    Science.gov (United States)

    Yang, Fan; Li, Lian; Liu, Huiling; Cai, Yafei; Wang, Genlin

    2012-04-01

    β-lactoglobulin (β-LG) is the major whey protein in the milk. In order to investigate the polymorphism of β-LG variant B precursor (β-LG B*: GenBank accession no. DQ489319) gene and its effects on the milk traits, the single-strand conformation polymorphism method (PCR-SSCP) were adopted to analyze polymorphism between 5229th and 5476th bp in the β-LG B* gene in Chinese Holstein. Four genotypes were found (AA, AB, AC and ABC) and 3 single nucleotide polymorphisms (SNPs) were detected (g.5239C>A, g.5240A>C, g.5305C>T and mix type g.5305C/T) in the exon 4 of β-LG B* gene. It was also found that the protein contents of AB, AC and ABC dairy cows were higher than AA (P A, g.5240A>C and g.5305C>T) might affect the milk trait and all of them were high polymorphism (0.5 Glu, Thr>Pro and Ala>Val) respectively, and the spatial secondary and tertiary structure forecasting result also showed that single amino acid change influence protein spatial structure change in Chinese Holstein. Taken together, it is suggested that these SNPs change β-LG B* gene structure and expression. The polymorphism possibly holds the secret of milk protein and fat contents in the milk of Chinese Holstein.

  8. Molecular Characterization of an Ice Nucleation Protein Variant (InaQ from Pseudomonas syringae and the Analysis of Its Transmembrane Transport Activity in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Qianqian Li, Qi Yan, Jinsi Chen, Yan He, Jing Wang, Hongxing Zhang, Ziniu Yu, Lin Li

    2012-01-01

    Full Text Available The ice nucleation protein (INP of Pseudomonas syringae has gained scientific interest not only because of its pathogenicity of foliar necroses but also for its wide range of potential applications, such as in snow making, frozen food preparation, and surface-display system development. However, studies on the transport activity of INP remain lacking. In the present study, a newly identified INP-gene variant, inaQ, from a P. syringae MB03 strain was cloned. Its structural domains, signal sequences, and the hydrophilicity or hydrophobicity of each domain, were then characterized. The deduced amino acid sequence of InaQ shares similar protein domains with three P. syringae INPs, namely, InaK, InaZ, and InaV, which were identified as an N-terminal domain, a central repeating domain, and a C-terminal domain. The expression of the full-length InaQ and of various truncated variants was induced in Escherichia coli to analyze their transmembrane transport and surface-binding activities, while using the green fluorescence protein (GFP as the fusion partner. With two transmembrane segments and a weak secretion signal, the N-terminal domain (InaQ-N alone was found to be responsible for the transport process as well as for the binding to the outer membrane, whereas the C-terminal region was nonfunctional in protein transport. Increased membrane transport and surface-binding capacities were induced by a low isopropyl-β-D-thiogalactoside concentration (0.1 mmol/l but not by culture temperatures (15 ºC to 37 ºC. Furthermore, by constructing the GFP-fused proteins with a single InaQ-N, as well as two and three tandemly aligned InaQ-N molecules, the transport and membrane-binding activities of these proteins were compared using Western blot analysis, immmunofluorescence microscopy, and assays of the GFP specific fluorescence intensity of subcellular fractions and flow cytometry, which showed that the increase of InaQ-N repeats resulted in a coordinated

  9. Molecular characterization of an ice nucleation protein variant (inaQ) from Pseudomonas syringae and the analysis of its transmembrane transport activity in Escherichia coli.

    Science.gov (United States)

    Li, Qianqian; Yan, Qi; Chen, Jinsi; He, Yan; Wang, Jing; Zhang, Hongxing; Yu, Ziniu; Li, Lin

    2012-01-01

    The ice nucleation protein (INP) of Pseudomonas syringae has gained scientific interest not only because of its pathogenicity of foliar necroses but also for its wide range of potential applications, such as in snow making, frozen food preparation, and surface-display system development. However, studies on the transport activity of INP remain lacking. In the present study, a newly identified INP-gene variant, inaQ, from a P. syringae MB03 strain was cloned. Its structural domains, signal sequences, and the hydrophilicity or hydrophobicity of each domain, were then characterized. The deduced amino acid sequence of InaQ shares similar protein domains with three P. syringae INPs, namely, InaK, InaZ, and InaV, which were identified as an N-terminal domain, a central repeating domain, and a C-terminal domain. The expression of the full-length InaQ and of various truncated variants was induced in Escherichia coli to analyze their transmembrane transport and surface-binding activities, while using the green fluorescence protein (GFP) as the fusion partner. With two transmembrane segments and a weak secretion signal, the N-terminal domain (InaQ-N) alone was found to be responsible for the transport process as well as for the binding to the outer membrane, whereas the C-terminal region was nonfunctional in protein transport. Increased membrane transport and surface-binding capacities were induced by a low isopropyl-β-D-thiogalactoside concentration (0.1 mmol/l) but not by culture temperatures (15 ºC to 37 ºC). Furthermore, by constructing the GFP-fused proteins with a single InaQ-N, as well as two and three tandemly aligned InaQ-N molecules, the transport and membrane-binding activities of these proteins were compared using Western blot analysis, immmunofluorescence microscopy, and assays of the GFP specific fluorescence intensity of subcellular fractions and flow cytometry, which showed that the increase of InaQ-N repeats resulted in a coordinated increase of the

  10. Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

    Directory of Open Access Journals (Sweden)

    Davidson Michael W

    2008-03-01

    Full Text Available Abstract Background In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP, the expanding set of fluorescent protein (FP variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1, a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra. Results Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His. Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP. To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins. Conclusion Based on the results of our

  11. Variants of Rab GTPase-Effector Binding Protein-2 Cause Variation in the Collateral Circulation and Severity of Stroke.

    Science.gov (United States)

    Lucitti, Jennifer L; Sealock, Robert; Buckley, Brian K; Zhang, Hua; Xiao, Lin; Dudley, Andrew C; Faber, James E

    2016-12-01

    The extent (number and diameter) of collateral vessels varies widely and is a major determinant, along with arteriogenesis (collateral remodeling), of variation in severity of tissue injury after large artery occlusion. Differences in genetic background underlie the majority of the variation in collateral extent in mice, through alterations in collaterogenesis (embryonic collateral formation). In brain and other tissues, ≈80% of the variation in collateral extent among different mouse strains has been linked to a region on chromosome 7. We recently used congenic (CNG) fine mapping of C57BL/6 (B6, high extent) and BALB/cByJ (BC, low extent) mice to narrow the region to a 737 Kb locus, Dce1. Herein, we report the causal gene. We used additional CNG mapping and knockout mice to narrow the number of candidate genes. Subsequent inspection identified a nonsynonymous single nucleotide polymorphism between B6 and BC within Rabep2 (rs33080487). We then created B6 mice with the BC single nucleotide polymorphism at this locus plus 3 other lines for predicted alteration or knockout of Rabep2 using gene editing. The single amino acid change caused by rs33080487 accounted for the difference in collateral extent and infarct volume between B6 and BC mice attributable to Dce1. Mechanistically, variants of Rabep2 altered collaterogenesis during embryogenesis but had no effect on angiogenesis examined in vivo and in vitro. Rabep2 deficiency altered endosome trafficking known to be involved in VEGF-A→VEGFR2 signaling required for collaterogenesis. Naturally occurring variants of Rabep2 are major determinants of variation in collateral extent and stroke severity in mice. © 2016 American Heart Association, Inc.

  12. Resistance to chronic wasting disease in transgenic mice expressing a naturally occurring allelic variant of deer prion protein

    NARCIS (Netherlands)

    Meade-White, K.; Race, B.; Trifilo, M.; Bossers, A.; Favara, C.; Lacasse, R.; Miller, M.; Williams, E.; Oldstone, M.; Race, R.; Chesebro, B.

    2007-01-01

    Prion protein (PrP) is a required factor for susceptibility to transmissible spongiform encephalopathy or prion diseases. In transgenic mice, expression of prion protein (PrP) from another species often confers susceptibility to prion disease from that donor species. For example, expression of deer

  13. Alternative Splicing Generates Different Parkin Protein Isoforms: Evidences in Human, Rat, and Mouse Brain

    Directory of Open Access Journals (Sweden)

    Soraya Scuderi

    2014-01-01

    Full Text Available Parkinson protein 2, E3 ubiquitin protein ligase (PARK2 gene mutations are the most frequent causes of autosomal recessive early onset Parkinson’s disease and juvenile Parkinson disease. Parkin deficiency has also been linked to other human pathologies, for example, sporadic Parkinson disease, Alzheimer disease, autism, and cancer. PARK2 primary transcript undergoes an extensive alternative splicing, which enhances transcriptomic diversification. To date several PARK2 splice variants have been identified; however, the expression and distribution of parkin isoforms have not been deeply investigated yet. Here, the currently known PARK2 gene transcripts and relative predicted encoded proteins in human, rat, and mouse are reviewed. By analyzing the literature, we highlight the existing data showing the presence of multiple parkin isoforms in the brain. Their expression emerges from conflicting results regarding the electrophoretic mobility of the protein, but it is also assumed from discrepant observations on the cellular and tissue distribution of parkin. Although the characterization of each predicted isoforms is complex, since they often diverge only for few amino acids, analysis of their expression patterns in the brain might account for the different pathogenetic effects linked to PARK2 gene mutations.

  14. Progesterone receptor (PR) variants exist in breast cancer cells characterised as PR negative.

    Science.gov (United States)

    Cork, David M W; Lennard, Thomas W J; Tyson-Capper, Alison J

    2012-12-01

    Progesterone receptor (PR) expression is measured in breast cancer by immunohistochemistry using N-terminally targeted antibodies and serves as a biomarker for endocrine therapeutic decisions. Extensive PR alternative splicing has been reported which may generate truncated PR variant proteins which are not detected by current breast cancer screening or may alter the function of proteins detected in screening. However, the existence of such truncated PR variants remains controversial. We have characterised PR protein expression in breast cancer cell lines using commercial PR antibodies targeting different epitopes. Truncated PR proteins are detected in reportedly PR negative MDA-MB-231 cells using a C-terminally targeted antibody. Antibody specificity was confirmed by immunoblotting following siRNA knockdown of PR expression. We have further demonstrated that alternatively spliced PR mRNA is present in MDA-MB-231 cells and in reportedly PR-negative breast tumour tissue which could encode the truncated PR proteins detected by the C-terminal antibody. The potential function of PR variant proteins present in MDA-MB-231 cells was also assessed, indicating the ability of these PR variants to bind progesterone, interact with a nuclear PR co-factor and bind DNA. These findings suggest that alternative splicing may generate functional truncated PR variant proteins which are not detected by breast cancer screening using N-terminally targeted antibodies leading to misclassification as PR negative.

  15. Calorimetric and spectroscopic properties of small globular proteins (bovine serum albumin, hemoglobin) after free radical generation

    Energy Technology Data Exchange (ETDEWEB)

    Farkas, N.; Belagyi, J.; Lorinczy, D

    2003-09-04

    Mild oxidation of -SH-containing proteins (serum albumin, hemoglobin) by Ce(IV)-ions in the presence of the spin trap phenyl-tert-butylnitrone (PBN) resulted in the appearance of strongly immobilized nitroxide free radicals which evidences the formation of thiyl radicals on the thiol site of the proteins. In hydroxyl free radical generating system a fraction of strongly immobilized nitroxide radicals was also detected in these proteins, which implies that the oxidation of a fraction of the thiol groups was also involved in the free radical reaction. According to the differential scanning calorimetry (DSC) experiments the melting processes of the proteins were calorimetrically irreversible, therefore the two-state kinetic model was used to evaluate the experiments. The results support the view that site-specific interaction of SH-containing proteins with hydroxyl and thiyl free radicals is able to modify the internal dynamics of proteins and affect the conformation of large molecules.

  16. Comparison of the aggregation of homologous β2-microglobulin variants reveals protein solubility as a key determinant of amyloid formation.

    Science.gov (United States)

    Pashley, Clare L; Hewitt, Eric W; Radford, Sheena E

    2016-02-13

    The mouse and human β2-microglobulin protein orthologs are 70% identical in sequence and share 88% sequence similarity. These proteins are predicted by various algorithms to have similar aggregation and amyloid propensities. However, whilst human β2m (hβ2m) forms amyloid-like fibrils in denaturing conditions (e.g. pH2.5) in the absence of NaCl, mouse β2m (mβ2m) requires the addition of 0.3M NaCl to cause fibrillation. Here, the factors which give rise to this difference in amyloid propensity are investigated. We utilise structural and mutational analyses, fibril growth kinetics and solubility measurements under a range of pH and salt conditions, to determine why these two proteins have different amyloid propensities. The results show that, although other factors influence the fibril growth kinetics, a striking difference in the solubility of the proteins is a key determinant of the different amyloidogenicity of hβ2m and mβ2m. The relationship between protein solubility and lag time of amyloid formation is not captured by current aggregation or amyloid prediction algorithms, indicating a need to better understand the role of solubility on the lag time of amyloid formation. The results demonstrate the key contribution of protein solubility in determining amyloid propensity and lag time of amyloid formation, highlighting how small differences in protein sequence can have dramatic effects on amyloid formation.

  17. Oxidation of myosin by haem proteins generates myosin radicals and protein cross-links

    DEFF Research Database (Denmark)

    Lametsch, Marianne Lund; Luxford, Catherine; Skibsted, Leif Horsfelt

    2008-01-01

    as a result of the reaction with activated haem proteins (horseradish peroxidase/H2O2) and met-myoglobin/H2O2) has been investigated by EPR spectroscopy and amino-acid consumption, product formation has been characterized by HPLC, and changes in protein integrity have been determined by SDS/PAGE. Multiple...... of thiyl and tyrosyl radicals is consistent with the observed consumption of cysteine and tyrosine residues, the detection of di-tyrosine by HPLC and the detection of both reducible (disulfide bond) and non-reducible cross-links between myosin molecules by SDS/PAGE. The time course of radical formation...

  18. Advanced oxidation protein products are generated by bovine neutrophils and inhibit free radical production in vitro.

    Science.gov (United States)

    Bordignon, Milena; Da Dalt, Laura; Marinelli, Lieta; Gabai, Gianfranco

    2014-01-01

    Despite the recognised importance of oxidative stress in the health and immune function of dairy cows, protein oxidation markers have been poorly studied in this species. The current study aimed to characterise markers of protein oxidation generated by activated bovine neutrophils and investigate the biological effects of advanced oxidation protein products (AOPP) on bovine neutrophils. Markers of protein oxidation (AOPP, dityrosines and carbonyls) were measured in culture medium containing bovine serum albumin (BSA) exposed to neutrophils. The effect of AOPP-BSA on generation of reactive oxygen species (ROS) was assessed by chemiluminescence. Activation of caspases-3, -8 and -9 and the presence of DNA laddering were used as apoptosis markers. Greater amounts of AOPP were generated by phorbol myristate acetate (PMA)-activated than non-activated neutrophils (1.46 ± 0.13 vs. 0.75 ± 0.13 nmol/mg protein, respectively; P<0.05). Activated neutrophils and hypochlorous acid generated slightly different patterns of oxidized protein markers. Exposure to AOPP-BSA did not stimulate ROS production. Activated neutrophils generated a lesser amount of ROS when incubated with AOPP-BSA (P<0.001). Activation with PMA induced a loss of viable neutrophils after 3h, which was greater with AOPP-BSA incubation (P<0.05). Detectable amounts of active caspases-3, -8 and -9 were found in nearly all samples but differences in caspase activation or DNA laddering were not observed comparing treatment groups. Apoptosis was unlikely to be responsible for the greater loss of PMA-activated neutrophils cultured in AOPP-BSA and it is possible that primary necrosis occurred. The results suggest that accumulation of oxidized proteins at an inflammatory site might result in a progressive reduction of neutrophil viability.

  19. A Functional Toll-Interacting Protein Variant Is Associated with Bacillus Calmette-Guérin-Specific Immune Responses and Tuberculosis.

    Science.gov (United States)

    Shah, Javeed A; Musvosvi, Munyaradzi; Shey, Muki; Horne, David J; Wells, Richard D; Peterson, Glenna J; Cox, Jeffery S; Daya, Michelle; Hoal, Eileen G; Lin, Lin; Gottardo, Raphael; Hanekom, Willem A; Scriba, Thomas J; Hatherill, Mark; Hawn, Thomas R

    2017-08-15

    The molecular mechanisms that regulate tuberculosis susceptibility and bacillus Calmette-Guérin (BCG)-induced immunity are mostly unknown. However, induction of the adaptive immune response is a critical step in host control of Mycobacterium tuberculosis. Toll-interacting protein (TOLLIP) is a ubiquitin-binding protein that regulates innate immune responses, including Toll-like receptor signaling, which initiate adaptive immunity. TOLLIP variation is associated with susceptibility to tuberculosis, but the mechanism by which it regulates tuberculosis immunity is poorly understood. To identify functional TOLLIP variants and evaluate the role of TOLLIP variation on innate and adaptive immune responses to mycobacteria and susceptibility to tuberculosis. We used human cellular immunology approaches to characterize the role of a functional TOLLIP variant on monocyte mRNA expression and M. tuberculosis-induced monocyte immune functions. We also examined the association of TOLLIP variation with BCG-induced T-cell responses and susceptibility to latent tuberculosis infection. We identified a functional TOLLIP promoter region single-nucleotide polymorphism, rs5743854, which was associated with decreased TOLLIP mRNA expression in infant monocytes. After M. tuberculosis infection, TOLLIP-deficient monocytes demonstrated increased IL-6, increased nitrite, and decreased bacterial replication. The TOLLIP-deficiency G/G genotype was associated with decreased BCG-specific IL-2(+) CD4(+) T-cell frequency and proliferation. This genotype was also associated with increased susceptibility to latent tuberculosis infection. TOLLIP deficiency is associated with decreased BCG-specific T-cell responses and increased susceptibility to tuberculosis. We hypothesize that the heightened antibacterial monocyte responses after vaccination of TOLLIP-deficient infants are responsible for decreased BCG-specific T-cell responses. Activating TOLLIP may provide a novel adjuvant strategy for BCG

  20. Comparative analysis of oncogenic properties and nuclear factor-kappaB activity of latent membrane protein 1 natural variants from Hodgkin's lymphoma's Reed-Sternberg cells and normal B-lymphocytes.

    Science.gov (United States)

    Faumont, Nathalie; Chanut, Aurélie; Benard, Alan; Cogne, Nadine; Delsol, Georges; Feuillard, Jean; Meggetto, Fabienne

    2009-03-01

    In Epstein-Barr virus-associated Hodgkin's lymphomas, neoplastic Reed-Sternberg cells and surrounding non-tumor B-cells contain different variants of the LMP1-BNLF1 oncogene. In this study, we raised the question of functional properties of latent membrane protein 1 (LMP1) natural variants from both Reed-Sternberg and non-tumor B-cells. Twelve LMP1 natural variants from Reed-Sternberg cells, non-tumor B-cells of Hodgkin's lymphomas and from B-cells of benign reactive lymph nodes were cloned, sequenced and stably transfected in murine recombinant interleukin-3-dependent Ba/F3 cells to search for relationships between LMP1 cellular origin and oncogenic properties as well as nuclear factor-kappaB activation, and apoptosis protection. LMP1 variants of Reed-Sternberg cell origin were often associated with increased mutation rate and with recurrent genetic events, such as del15bp associated with S to N replacement at codon 309, and four substitutions I85L, F106Y, I122L, and M129I. Oncogenic potential (growth factor-independence plus clonogenicity) was consistently associated with LMP1 variants from Reed-Sternberg cells, but inconstantly for LMP1-variants from non-tumor B-cells. Analysis of LMP1 variants from both normal B-cells and Reed-Sternberg cells indicates that protection against apoptosis through activation of nuclear factor-kappaB - whatever the cellular origin of LMP1 - was maintained intact, regardless of the mutational pattern. Taken together, our results demonstrate that preserved nuclear factor-kappaB activity and protection against apoptosis would be the minimal prerequisites for all LMP1 natural variants from both normal and tumor cells in Hodgkin's lymphomas, and that oncogenic potential would constitute an additional feature for LMP1 natural variants in Reed-Sternberg cells.

  1. Rare, Non-Synonymous Variant in the Smooth Muscle-Specific Isoform of Myosin Heavy Chain, MYH11, R247C, Alters Force Generation in the Aorta and Phenotype of Smooth Muscle Cells

    Science.gov (United States)

    Kuang, Shao-Qing; Kwartler, Callie S.; Byanova, Katerina L.; Pham, John; Gong, Limin; Prakash, Siddharth K.; Huang, Jian; Kamm, Kristine E.; Stull, James T.; Sweeney, H. Lee; Milewicz, Dianna M.

    2013-01-01

    Rationale Mutations in MYH11 cause autosomal dominant inheritance of thoracic aortic aneurysms and dissections. At the same time, rare, non-synonymous variants in MYH11 that are predicted to disrupt protein function but do not cause inherited aortic disease are common in the general population and the vascular disease risk associated with these variants is unknown. Objective To determine the consequences of the recurrent MYH11 rare variant, R247C, through functional studies in vitro and analysis of a knock-in mouse model with this specific variant, including assessment of aortic contraction, response to vascular injury, and phenotype of primary aortic smooth muscle cells (SMCs). Methods and Results The steady state ATPase activity (actin-activated) and the rates of phosphate and ADP release were lower for the R247C mutant myosin than for the wild-type, as was the rate of actin filament sliding in an in vitro motility assay. Myh11R247C/R247C mice exhibited normal growth, reproduction, and aortic histology but decreased aortic contraction. In response to vascular injury, Myh11R247C/R247C mice showed significantly increased neointimal formation due to increased SMC proliferation when compared with the wild-type mice. Primary aortic SMCs explanted from the Myh11R247C/R247C mice were de-differentiated compared with wild-type SMCs based on increased proliferation and reduced expression of SMC contractile proteins. The mutant SMCs also displayed altered focal adhesions and decreased Rho activation, associated with decreased nuclear localization of myocardin-related transcription factor-A. Exposure of the Myh11R247C/R247C SMCs to a Rho activator rescued the de-differentiated phenotype of the SMCs. Conclusions These results indicate that a rare variant in MYH11, R247C, alters myosin contractile function and SMC phenotype, leading to increased proliferation in vitro and in response to vascular injury. PMID:22511748

  2. αB-Crystallin R120G variant causes cardiac arrhythmias and alterations in the expression of Ca(2+) -handling proteins and endoplasmic reticulum stress in mice.

    Science.gov (United States)

    Jiao, Qibin; Sanbe, Atsushi; Zhang, Xingwei; Liu, Jun-Ping; Minamisawa, Susumu

    2014-08-01

    Mutations of αB-crystallin (CryαB), a small heat shock protein abundantly expressed in cardiac and skeletal muscles, are known to cause desmin-related myopathies. The CryαB R120G allele has been linked to a familial desminopathy and, in transgenic mice, causes a sudden death at about 28 weeks of age. To investigate the mechanisms of the sudden cardiac arrest of CryαB R120G transgenic mice, we prepared protein samples from left ventricular tissues of two different age groups (10 and 28 weeks) and examined Ca(2+) -handling proteins. Expression of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) 2, phospholamban, ryanodine receptor 2 and calsequestrin 2 was significantly decreased in 28- versus 10-week-old CryαB R120G transgenic mice. In addition, low heart rate variability, including heart rate, total power and low frequency, was observed and continuous electrocardiogram monitoring revealed cardiac arrhythmias, such as ventricular tachycardia, atrioventricular block and atrial flutter, in 28-week-old CryαB R120G transgenic mice. In contrast, expression of endoplasmic reticulum (ER) degradation enhancing α-mannosidase-like protein, inositol requirement 1 and X-box binding protein 1 were increased significantly in 28- versus 10-week-old CryαBR120G transgenic mice, suggesting that the CryαBR120G transgenic mice exhibit increased ER stress compared with wild-type mice. Together, the data suggest that the CryαB R120G dominant variant induces ER stress and impairs Ca(2+) regulation, leading to ageing-related cardiac dysfunction, arrhythmias and decreased autonomic tone with shortened lifespan.

  3. Generation and Characterization of a Monoclonal Antibody Against prM Protein of West Nile Virus

    OpenAIRE

    Guo, Li-Ping; Huo, Hong; Wang, Xiao-Lei; Bu, Zhi-Gao; Hua, Rong-Hong

    2014-01-01

    West Nile virus (WNV), which is an emerging pathogenic flavivirus with increasing distribution worldwide, is the cause of major human and animal health concerns. The pre-membrane (prM) protein of WNV is cleaved during maturation by the furin protease into the structural protein M and a pr-segment. In this study we generated and characterized a monoclonal antibody (MAb) against the WNV prM protein. Western blot analysis showed that the MAb reacted with WNV prM specifically. Immunohistochemistr...

  4. Protein Z efficiently depletes thrombin generation in disseminated intravascular coagulation with poor prognosis.

    Science.gov (United States)

    Lee, Nuri; Kim, Ji-Eun; Gu, Ja-Yoon; Yoo, Hyun Ju; Kim, Inho; Yoon, Sung-Soo; Park, Seonyang; Han, Kyou-Sup; Kim, Hyun Kyung

    2016-01-01

    Disseminated intravascular coagulation (DIC) is characterized by consumption of coagulation factors and anticoagulants. Thrombin generation assay (TGA) gives useful information about global hemostatic status. We developed a new TGA system that anticoagulant addition can deplete thrombin generation in plasma, which may reflect defective anticoagulant system in DIC. TGAs were measured on the calibrated automated thrombogram with and without thrombomodulin or protein Z in 152 patients who were suspected of having DIC, yielding four parameters including lag time, endogenous thrombin potential, peak thrombin and time-to-peak in each experiment. Nonsurvivors showed significantly prolonged lag time and time-to-peak in TGA-protein Z system, which was performed with added protein Z. In multivariate Cox regression analysis, lag time and time-to-peak in TGA system were significant independent prognostic factors. In TGA-protein Z system, lag time and time-to-peak were revealed as independent prognostic factors of DIC. Protein Z addition could potentiate its anticoagulant effect in DIC with poor prognosis, suggesting the presence of defective protein Z system. The prolonged lag time and time-to-peak in both TGA and TGA-protein Z systems are expected to be used as independent prognostic factors of DIC.

  5. Identification of an immunogenic protein of Giardia lamblia using monoclonal antibodies generated from infected mice

    Directory of Open Access Journals (Sweden)

    Jael Quintero

    2013-08-01

    Full Text Available The humoral immune response plays an important role in the clearance of Giardia lamblia. However, our knowledge about the specific antigens of G. lamblia that induce a protective immune response is limited. The purpose of this study was to identify and characterise the immunogenic proteins of G. lamblia in a mouse model. We generated monoclonal antibodies (moAbs specific to G. lamblia (1B10, 2C9.D11, 3C10.E5, 3D10, 5G8.B5, 5F4, 4C7, 3C5 and 3C6 by fusing splenocytes derived from infected mice. Most of these moAbs recognised a band of ± 71 kDa (5G8 protein and this protein was also recognised by serum from the infected mice. We found that the moAbs recognised conformational epitopes of the 5G8 protein and that this antigen is expressed on the cell surface and inside trophozoites. Additionally, antibodies specific to the 5G8 protein induced strong agglutination (> 70-90% of trophozoites. We have thus identified a highly immunogenic antigen of G. lamblia that is recognised by the immune system of infected mice. In summary, this study describes the identification and partial characterisation of an immunogenic protein of G. lamblia. Additionally, we generated a panel of moAbs specific for this protein that will be useful for the biochemical and immunological characterisation of this immunologically interesting Giardia molecule.

  6. Molecular interactions of proteins and peptides at interfaces studied by sum frequency generation vibrational spectroscopy.

    Science.gov (United States)

    Liu, Yuwei; Jasensky, Joshua; Chen, Zhan

    2012-01-31

    Interfacial peptides and proteins are critical in many biological processes and thus are of interest to various research fields. To study these processes, surface sensitive techniques are required to completely describe different interfacial interactions intrinsic to many complicated processes. Sum frequency generation (SFG) spectroscopy has been developed into a powerful tool to investigate these interactions and mechanisms of a variety of interfacial peptides and proteins. It has been shown that SFG has intrinsic surface sensitivity and the ability to acquire conformation, orientation, and ordering information about these systems. This paper reviews recent studies on peptide/protein-substrate interactions, peptide/protein-membrane interactions, and protein complexes at interfaces and demonstrates the ability of SFG on unveiling the molecular pictures of complicated interfacial biological processes. © 2011 American Chemical Society

  7. Cost of unneeded proteins in E. coli is reduced after several generations in exponential growth.

    Science.gov (United States)

    Shachrai, Irit; Zaslaver, Alon; Alon, Uri; Dekel, Erez

    2010-06-11

    When E. coli cells express unneeded protein, they grow more slowly. Such penalty to fitness associated with making proteins is called protein cost. Protein cost is an important component in the cost-benefit tradeoffs that underlie the evolution of protein circuits, but its origins are still poorly understood. Here, we ask how the protein cost varies during the exponential growth phase of E. coli. We find that cells growing exponentially following an upshift from overnight culture show a large cost when producing unneeded proteins. However, after several generations, while still in exponential growth, the cells enter a phase where cost is much reduced despite vigorous unneeded protein production. We find that this reduced-cost phase depends on the ppGpp system, which adjusts the amount of ribosomes in the cell and does not occur after a downshift from rich to poor medium. These findings suggest that protein cost is a transient phenomenon that happens upon an upshift in conditions and that cost is reduced when ribosomes and other cellular systems have increased to their appropriate steady-state level in the new condition.

  8. Contribution of factor H-Binding protein sequence to the cross-reactivity of meningococcal native outer membrane vesicle vaccines with over-expressed fHbp variant group 1.

    Science.gov (United States)

    Marini, Arianna; Rossi, Omar; Aruta, Maria Grazia; Micoli, Francesca; Rondini, Simona; Guadagnuolo, Serafina; Delany, Isabel; Henderson, Ian R; Cunningham, Adam F; Saul, Allan; MacLennan, Calman A; Koeberling, Oliver

    2017-01-01

    Factor H-binding protein (fHbp) is an important meningococcal vaccine antigen. Native outer membrane vesicles with over-expressed fHbp (NOMV OE fHbp) have been shown to induce antibodies with broader functional activity than recombinant fHbp (rfHbp). Improved understanding of this broad coverage would facilitate rational vaccine design. We performed a pair-wise analysis of 48 surface-exposed amino acids involved in interacting with factor H, among 383 fHbp variant group 1 sequences. We generated isogenic NOMV-producing meningococcal strains from an African serogroup W isolate, each over-expressing one of four fHbp variant group 1 sequences (ID 1, 5, 9, or 74), including those most common among invasive African meningococcal isolates. Mice were immunised with each NOMV, and sera tested for IgG levels against each of the rfHbp ID and for ability to kill a panel of heterologous meningococcal isolates. At the fH-binding site, ID pairs differed by a maximum of 13 (27%) amino acids. ID 9 shared an amino acid sequence common to 83 ID types. The selected ID types differed by up to 6 amino acids, in the fH-binding site. All NOMV and rfHbp induced high IgG levels against each rfHbp. Serum killing from mice immunised with rfHbp was generally less efficient and more restricted compared to NOMV, which induced antibodies that killed most meningococci tested, with decreased stringency for ID type differences. Breadth of killing was mostly due to anti-fHbp antibodies, with some restriction according to ID type sequence differences. Nevertheless, under our experimental conditions, no relationship between antibody cross-reactivity and variation fH-binding site sequence was identified. NOMV over-expressing different fHbp IDs belonging to variant group 1 induce antibodies with fine specificities against fHbp, and ability to kill broadly meningococci expressing heterologous fHbp IDs. The work reinforces that meningococcal NOMV with OE fHbp is a promising vaccine strategy, and provides

  9. Insertion of Cecropin A and reconstitution of bacterial outer membrane protein FhuA variants in polymeric membranes

    OpenAIRE

    Muhammad, Noor

    2011-01-01

    Polymer based nanocompartments (polymersomes) have potential applications in synthetic biology (pathway engineering), medicine (drug release), and industrial biotechnology (chiral nanoreactors, multistep synthesis, bioconversions in non-aqueous environments, and selective product recovery). The aforementioned goals can be accomplished by polymer membrane functionalization through covalent bonding or inclusion of proteins/peptides, to obtain specific properties like recognition, catalytic acti...

  10. Usefulness of a Darwinian system in a biotechnological application: evolution of optical window fluorescent protein variants under selective pressure.

    Science.gov (United States)

    Schoetz, Ulrike; Deliolanis, Nikolaos C; Ng, David; Pauli, Jutta; Resch-Genger, Ute; Kühn, Enrico; Heuer, Steffen; Beisker, Wolfgang; Köster, Reinhard W; Zitzelsberger, Horst; Caldwell, Randolph B

    2014-01-01

    With rare exceptions, natural evolution is an extremely slow process. One particularly striking exception in the case of protein evolution is in the natural production of antibodies. Developing B cells activate and diversify their immunoglobulin (Ig) genes by recombination, gene conversion (GC) and somatic hypermutation (SHM). Iterative cycles of hypermutation and selection continue until antibodies of high antigen binding specificity emerge (affinity maturation). The avian B cell line DT40, a cell line which is highly amenable to genetic manipulation and exhibits a high rate of targeted integration, utilizes both GC and SHM. Targeting the DT40's diversification machinery onto transgenes of interest inserted into the Ig loci and coupling selective pressure based on the desired outcome mimics evolution. Here we further demonstrate the usefulness of this platform technology by selectively pressuring a large shift in the spectral properties of the fluorescent protein eqFP615 into the highly stable and advanced optical imaging expediting fluorescent protein Amrose. The method is advantageous as it is time and cost effective and no prior knowledge of the outcome protein's structure is necessary. Amrose was evolved to have high excitation at 633 nm and excitation/emission into the far-red, which is optimal for whole-body and deep tissue imaging as we demonstrate in the zebrafish and mouse model.

  11. Usefulness of a Darwinian system in a biotechnological application: evolution of optical window fluorescent protein variants under selective pressure.

    Directory of Open Access Journals (Sweden)

    Ulrike Schoetz

    Full Text Available With rare exceptions, natural evolution is an extremely slow process. One particularly striking exception in the case of protein evolution is in the natural production of antibodies. Developing B cells activate and diversify their immunoglobulin (Ig genes by recombination, gene conversion (GC and somatic hypermutation (SHM. Iterative cycles of hypermutation and selection continue until antibodies of high antigen binding specificity emerge (affinity maturation. The avian B cell line DT40, a cell line which is highly amenable to genetic manipulation and exhibits a high rate of targeted integration, utilizes both GC and SHM. Targeting the DT40's diversification machinery onto transgenes of interest inserted into the Ig loci and coupling selective pressure based on the desired outcome mimics evolution. Here we further demonstrate the usefulness of this platform technology by selectively pressuring a large shift in the spectral properties of the fluorescent protein eqFP615 into the highly stable and advanced optical imaging expediting fluorescent protein Amrose. The method is advantageous as it is time and cost effective and no prior knowledge of the outcome protein's structure is necessary. Amrose was evolved to have high excitation at 633 nm and excitation/emission into the far-red, which is optimal for whole-body and deep tissue imaging as we demonstrate in the zebrafish and mouse model.

  12. Broadband photon pair generation in green fluorescent proteins through spontaneous four-wave mixing.

    Science.gov (United States)

    Shi, Siyuan; Thomas, Abu; Corzo, Neil V; Kumar, Prem; Huang, Yuping; Lee, Kim Fook

    2016-04-14

    Recent studies in quantum biology suggest that quantum mechanics help us to explore quantum processes in biological system. Here, we demonstrate generation of photon pairs through spontaneous four-wave mixing process in naturally occurring fluorescent proteins. We develop a general empirical method for analyzing the relative strength of nonlinear optical interaction processes in five different organic fluorophores. Our results indicate that the generation of photon pairs in green fluorescent proteins is subject to less background noises than in other fluorophores, leading to a coincidence-to-accidental ratio ~145. As such proteins can be genetically engineered and fused to many biological cells, our experiment enables a new platform for quantum information processing in a biological environment such as biomimetic quantum networks and quantum sensors.

  13. Broadband photon pair generation in green fluorescent proteins through spontaneous four-wave mixing

    Science.gov (United States)

    Shi, Siyuan; Thomas, Abu; Corzo, Neil V.; Kumar, Prem; Huang, Yuping; Lee, Kim Fook

    2016-04-01

    Recent studies in quantum biology suggest that quantum mechanics help us to explore quantum processes in biological system. Here, we demonstrate generation of photon pairs through spontaneous four-wave mixing process in naturally occurring fluorescent proteins. We develop a general empirical method for analyzing the relative strength of nonlinear optical interaction processes in five different organic fluorophores. Our results indicate that the generation of photon pairs in green fluorescent proteins is subject to less background noises than in other fluorophores, leading to a coincidence-to-accidental ratio ~145. As such proteins can be genetically engineered and fused to many biological cells, our experiment enables a new platform for quantum information processing in a biological environment such as biomimetic quantum networks and quantum sensors.

  14. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  15. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  16. Genetic variants in insulin-like growth factor binding protein-3 are associated with prostate cancer susceptibility in Eastern Chinese Han men

    Directory of Open Access Journals (Sweden)

    Zhang G

    2015-12-01

    Full Text Available Guiming Zhang,1–3 Yao Zhu,1,2 Fang Liu,4,5 Chengyuan Gu,1,2 Haitao Chen,4,5 Jianfeng Xu,4–6 Dingwei Ye1,2 1Department of Urology, Fudan University Shanghai Cancer Center, 2Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 3Department of Urology, The Affiliated Hospital of Qingdao University, Shandong, 4Fudan Institute of Urology, Huashan Hospital, Fudan University, 5State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, People’s Republic of China; 6Center for Cancer Genomics, Wake Forest School of Medicine, Winston-Salem, NC, USA Background: Growing evidence has indicated that insulin-like growth factor binding protein-3 (IGFBP-3 polymorphisms are associated with altered risk of prostate cancer (PCa. However, few studies have been conducted in Chinese population to validate this association. Materials and methods: Herein, we examined the association between genetic variants in the IGFBP-3 gene and PCa risk in the Chinese Han population based on a genome-wide association study (1,417 cases and 1,008 controls, and replicated three genetic variants loci in an independent case-control study (1,755 cases and 1,523 controls using Sequenom platform. Logistic regression analyses were performed to estimate odds ratios (ORs and 95% confidence intervals (95% CIs. Results: We found that in the discovery stage, rs9691259 (OR =0.691, 95% CI: 0.587–0.814, P<0.001 and rs6950179 (OR =1.420, 95% CI: 1.201–1.677, P<0.001 were significantly associated with PCa risk, whereas rs2854744 showed a marginal association with PCa risk. In the replication stage, the association between rs9691259 and rs6950179 and PCa risk was not replicated, whereas rs2854744 conferred a significant association with PCa risk (OR =1.399, 95% CI: 1.010–1.937, P=0.043. After combining the two stages, we found that rs9691259, rs6950179, and rs2854744 were all significantly associated with PCa risk. Conclusion

  17. Are C-reactive protein associated genetic variants associated with serum levels and retinal markers of microvascular pathology in Asian populations from Singapore?

    Directory of Open Access Journals (Sweden)

    Rajkumar Dorajoo

    Full Text Available INTRODUCTION: C-reactive protein (CRP levels are associated with cardiovascular disease and systemic inflammation. We assessed whether CRP-associated loci were associated with serum CRP and retinal markers of microvascular disease, in Asian populations. METHODS: Genome-wide association analysis (GWAS for serum CRP was performed in East-Asian Chinese (N = 2,434 and Malays (N = 2,542 and South-Asian Indians (N = 2,538 from Singapore. Leveraging on GWAS data, we assessed, in silico, association levels among the Singaporean datasets for 22 recently identified CRP-associated loci. At loci where directional inconsistencies were observed, quantification of inter-ethnic linkage disequilibrium (LD difference was determined. Next, we assessed association for a variant at CRP and retinal vessel traits [central retinal artery equivalent (CRAE and central retinal vein equivalent (CRVE] in a total of 24,132 subjects of East-Asian, South-Asian and European ancestry. RESULTS: Serum CRP was associated with SNPs in/near APOE, CRP, HNF1A and LEPR (p-values ≤4.7×10(-8 after meta-analysis of Singaporean populations. Using a candidate-SNP approach, we further replicated SNPs at 4 additional loci that had been recently identified to be associated with serum CRP (IL6R, GCKR, IL6 and IL1F10 (p-values ≤0.009, in the Singaporean datasets. SNPs from these 8 loci explained 4.05% of variance in serum CRP. Two SNPs (rs2847281 and rs6901250 were detected to be significant (p-value ≤0.036 but with opposite effect directions in the Singaporean populations as compared to original European studies. At these loci we did not detect significant inter-population LD differences. We further did not observe a significant association between CRP variant and CRVE or CRAE levels after meta-analysis of all Singaporean and European datasets (p-value >0.058. CONCLUSIONS: Common variants associated with serum CRP, first detected in primarily European studies, are also

  18. Genetic association analysis of ATP binding cassette protein family reveals a novel association of ABCB1 genetic variants with epilepsy risk, but not with drug-resistance.

    Science.gov (United States)

    Balan, Shabeesh; Bharathan, Sumitha Prameela; Vellichiramal, Neetha Nanoth; Sathyan, Sanish; Joseph, Vijai; Radhakrishnan, Kurupath; Banerjee, Moinak

    2014-01-01

    Epilepsy constitutes a heterogeneous group of disorders that is characterized by recurrent unprovoked seizures due to widely different etiologies. Multidrug resistance remains a major issue in clinical epileptology, where one third of patients with epilepsy continue to have seizures. Role of efflux transporters in multidrug resistant epilepsy has been attributed to drug-resistant epilepsy although, with discrepant observation in genetic studies. These discrepancies could be attributed to variety of factors such as variable definition of the anti-epileptic drug (AED)-resistance, variable epilepsy phenotypes and ethnicities among the studies. In the present study we inquired the role of multidrug transporters ABCB1 and ABCG2 variants in determining AED-resistance and susceptibility to epilepsy in three well-characterized cohorts comprising of mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (prototype for AED-resistant epilepsy); juvenile myoclonic epilepsy (JME) (prototype for AED-responsive epilepsy); and healthy non-epileptic controls, in 738 subjects of Malayalam speaking south Indian ancestry. ABCB1 and ABCG2 variants were not found to be associated with drug resistance when AED-resistant and AED-responsive cohorts were compared. However, a significant association was observed between ABCB1 (C3435T) rs1045642 and risk of having epilepsy (MTLE-HS and JME pooled cohort; genotypic p-value = 0.0002; allelic p-value = 0.004). This association was seen persistent with MTLE-HS (genotypic p-value = 0.0008; allelic p-value = 0.004) and also with JME (genotypic p-value = 0.01; allelic p-value = 0.05) cohort individually. In-silico functional prediction indicated that ABCB1 rs1045642 has a deleterious impact on protein coding function and in splicing regulation. We conclude that the ABCB1 and ABCG2 variants do not confer to AED-resistance in the study population. However, ABCB1 rs1045642 increases vulnerability to epilepsy with greater tendency for MTLE

  19. Genetic association analysis of ATP binding cassette protein family reveals a novel association of ABCB1 genetic variants with epilepsy risk, but not with drug-resistance.

    Directory of Open Access Journals (Sweden)

    Shabeesh Balan

    Full Text Available Epilepsy constitutes a heterogeneous group of disorders that is characterized by recurrent unprovoked seizures due to widely different etiologies. Multidrug resistance remains a major issue in clinical epileptology, where one third of patients with epilepsy continue to have seizures. Role of efflux transporters in multidrug resistant epilepsy has been attributed to drug-resistant epilepsy although, with discrepant observation in genetic studies. These discrepancies could be attributed to variety of factors such as variable definition of the anti-epileptic drug (AED-resistance, variable epilepsy phenotypes and ethnicities among the studies. In the present study we inquired the role of multidrug transporters ABCB1 and ABCG2 variants in determining AED-resistance and susceptibility to epilepsy in three well-characterized cohorts comprising of mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS (prototype for AED-resistant epilepsy; juvenile myoclonic epilepsy (JME (prototype for AED-responsive epilepsy; and healthy non-epileptic controls, in 738 subjects of Malayalam speaking south Indian ancestry. ABCB1 and ABCG2 variants were not found to be associated with drug resistance when AED-resistant and AED-responsive cohorts were compared. However, a significant association was observed between ABCB1 (C3435T rs1045642 and risk of having epilepsy (MTLE-HS and JME pooled cohort; genotypic p-value = 0.0002; allelic p-value = 0.004. This association was seen persistent with MTLE-HS (genotypic p-value = 0.0008; allelic p-value = 0.004 and also with JME (genotypic p-value = 0.01; allelic p-value = 0.05 cohort individually. In-silico functional prediction indicated that ABCB1 rs1045642 has a deleterious impact on protein coding function and in splicing regulation. We conclude that the ABCB1 and ABCG2 variants do not confer to AED-resistance in the study population. However, ABCB1 rs1045642 increases vulnerability to epilepsy with greater tendency

  20. Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process

    Science.gov (United States)

    Iacovache, Ioan; de Carlo, Sacha; Cirauqui, Nuria; Dal Peraro, Matteo; van der Goot, F. Gisou; Zuber, Benoît

    2016-07-01

    Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.

  1. Fatty Acid Binding Protein-1 (FABP1) and the Human FABP1 T94A Variant: Roles in the Endocannabinoid System and Dyslipidemias.

    Science.gov (United States)

    Schroeder, Friedhelm; McIntosh, Avery L; Martin, Gregory G; Huang, Huan; Landrock, Danilo; Chung, Sarah; Landrock, Kerstin K; Dangott, Lawrence J; Li, Shengrong; Kaczocha, Martin; Murphy, Eric J; Atshaves, Barbara P; Kier, Ann B

    2016-06-01

    The first discovered member of the mammalian FABP family, liver fatty acid binding protein (FABP1, L-FABP), occurs at high cytosolic concentration in liver, intestine, and in the case of humans also in kidney. While the rat FABP1 is well studied, the extent these findings translate to human FABP1 is not clear-especially in view of recent studies showing that endocannabinoids and cannabinoids represent novel rat FABP1 ligands and FABP1 gene ablation impacts the hepatic endocannabinoid system, known to be involved in non-alcoholic fatty liver (NAFLD) development. Although not detectable in brain, FABP1 ablation nevertheless also impacts brain endocannabinoids. Despite overall tertiary structure similarity, human FABP1 differs significantly from rat FABP1 in secondary structure, much larger ligand binding cavity, and affinities/specificities for some ligands. Moreover, while both mouse and human FABP1 mediate ligand induction of peroxisome proliferator activated receptor-α (PPARα), they differ markedly in pattern of genes induced. This is critically important because a highly prevalent human single nucleotide polymorphism (SNP) (26-38 % minor allele frequency and 8.3 ± 1.9 % homozygous) results in a FABP1 T94A substitution that further accentuates these species differences. The human FABP1 T94A variant is associated with altered body mass index (BMI), clinical dyslipidemias (elevated plasma triglycerides and LDL cholesterol), atherothrombotic cerebral infarction, and non-alcoholic fatty liver disease (NAFLD). Resolving human FABP1 and the T94A variant's impact on the endocannabinoid and cannabinoid system is an exciting challenge due to the importance of this system in hepatic lipid accumulation as well as behavior, pain, inflammation, and satiety.

  2. Collaborative pooled analysis of data on C-reactive protein gene variants and coronary disease: judging causality by Mendelian randomisation

    DEFF Research Database (Denmark)

    Danesh, J.; Hingorani, A.; Wensley, F.

    2008-01-01

    Many prospective studies have reported associations between circulating C-reactive protein (CRP) levels and risk of coronary heart disease (CHD), but causality remains uncertain. Studies of CHD are being conducted that involve measurement of common polymorphisms of the CRP gene known to be associ...... of low-grade inflammation to CHD and indicate whether or not CRP itself is involved in long-term pathogenesis Udgivelsesdato: 2008...

  3. Counteraction of the Antiapoptotic Protein Survivin by Diverting Expression to its Proapoptotic Splice Variant Survivin-2B

    Science.gov (United States)

    2010-01-01

    negatively regulated by low glucose. [17] Further, glucose restriction activates the longevity- associated histone and protein deacetylase, SIRT1 ...pattern of decreasing with low glucose. We also studied SIRT1 because it was already reported to epigenetically silence survivin transcription and...furthermore to be upregulated during glucose restriction. Indeed, SIRT1 increased with glucose restriction, in opposite manner as survivin and survivin-2B

  4. Allelic variants of the amylose extender mutation of maize demonstrate phenotypic variation in starch structure resulting from modified protein–protein interactions

    Science.gov (United States)

    Liu, Fushan; Ahmed, Zaheer; Lee, Elizabeth A.; Donner, Elizabeth; Liu, Qiang; Ahmed, Regina; Morell, Matthew K.; Emes, Michael J.; Tetlow, Ian J.

    2012-01-01

    amylose extender (ae−) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae− maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein–protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae− mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272–Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16–20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-32P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the

  5. Characterization of Intra-Type Variants of Oncogenic Human Papillomaviruses by Next-Generation Deep Sequencing of the E6/E7 Region

    Directory of Open Access Journals (Sweden)

    Enrico Lavezzo

    2016-03-01

    Full Text Available Different human papillomavirus (HPV types are characterized by differences in tissue tropism and ability to promote cell proliferation and transformation. In addition, clinical and experimental studies have shown that some genetic variants/lineages of high-risk HPV (HR-HPV types are characterized by increased oncogenic activity and probability to induce cancer. In this study, we designed and validated a new method based on multiplex PCR-deep sequencing of the E6/E7 region of HR-HPV types to characterize HPV intra-type variants in clinical specimens. Validation experiments demonstrated that this method allowed reliable identification of the different lineages of oncogenic HPV types. Advantages of this method over other published methods were represented by its ability to detect variants of all HR-HPV types in a single reaction, to detect variants of HR-HPV types in clinical specimens with multiple infections, and, being based on sequencing of the full E6/E7 region, to detect amino acid changes in these oncogenes potentially associated with increased transforming activity.

  6. Characterization of Intra-Type Variants of Oncogenic Human Papillomaviruses by Next-Generation Deep Sequencing of the E6/E7 Region.

    Science.gov (United States)

    Lavezzo, Enrico; Masi, Giulia; Toppo, Stefano; Franchin, Elisa; Gazzola, Valentina; Sinigaglia, Alessandro; Masiero, Serena; Trevisan, Marta; Pagni, Silvana; Palù, Giorgio; Barzon, Luisa

    2016-03-14

    Different human papillomavirus (HPV) types are characterized by differences in tissue tropism and ability to promote cell proliferation and transformation. In addition, clinical and experimental studies have shown that some genetic variants/lineages of high-risk HPV (HR-HPV) types are characterized by increased oncogenic activity and probability to induce cancer. In this study, we designed and validated a new method based on multiplex PCR-deep sequencing of the E6/E7 region of HR-HPV types to characterize HPV intra-type variants in clinical specimens. Validation experiments demonstrated that this method allowed reliable identification of the different lineages of oncogenic HPV types. Advantages of this method over other published methods were represented by its ability to detect variants of all HR-HPV types in a single reaction, to detect variants of HR-HPV types in clinical specimens with multiple infections, and, being based on sequencing of the full E6/E7 region, to detect amino acid changes in these oncogenes potentially associated with increased transforming activity.

  7. Detection of TET2, KRAS and CBL variants by Next Generation Sequencing and analysis of their correlation with JAK2 and FLT3 in childhood AML

    Directory of Open Access Journals (Sweden)

    Dilara Fatma Akin

    2016-04-01

    Conclusion: We found novel mutations for TET2, KRAS, and CBL. The detected variants in this article seem to be the first screening results of genes studied by NGS in childhood AML patients. Our results also showed some degree of association between FLT3-ITD and TET2, KRAS, CBL mutations.

  8. Photo-oxidation of cells generates long-lived intracellular protein peroxides

    DEFF Research Database (Denmark)

    Wright, Adam; Hawkins, Clare Louise; Davies, Michael Jonathan

    2003-01-01

    Singlet oxygen is generated by several cellular, enzymatic, and chemical reactions as well as by exposure to UV or visible light in the presence of a sensitizer. Consequently, this oxidant has been proposed to be a damaging agent many pathologies. Proteins are major targets for singlet oxygen...... as a result of their abundance and high rate constants for reaction. In this study, we show that illumination of viable rose bengal-loaded THP-1 (human monocyte-like) cells with visible light gives rise to intracellular protein-derived peroxides. The peroxide yield increases with illumination time, requires....../2) about 4 h at 37 degrees C. Decomposition of protein peroxides formed within cells, or on isolated cellular proteins, by metal ions gives rise to radicals as detected by EPR spin trapping. These studies demonstrate that exposure of intact cells to visible light in the presence of a sensitizer leads...

  9. Transcription Factor RUNX1 Regulates Platelet PCTP (Phosphatidylcholine Transfer Protein): Implications for Cardiovascular Events: Differential Effects of RUNX1 Variants.

    Science.gov (United States)

    Mao, Guangfen; Songdej, Natthapol; Voora, Deepak; Goldfinger, Lawrence E; Del Carpio-Cano, Fabiola E; Myers, Rachel A; Rao, A Koneti

    2017-09-05

    PCTP (phosphatidylcholine transfer protein) regulates the intermembrane transfer of phosphatidylcholine. Higher platelet PCTP expression is associated with increased platelet responses on activation of protease-activated receptor 4 thrombin receptors noted in black subjects compared with white subjects. Little is known about the regulation of platelet PCTP. Haplodeficiency of RUNX1, a major hematopoietic transcription factor, is associated with thrombocytopenia and impaired platelet responses on activation. Platelet expression profiling of a patient with a RUNX1 loss-of-function mutation revealed a 10-fold downregulation of the PCTP gene compared with healthy controls. We pursued the hypothesis that PCTP is regulated by RUNX1 and that PCTP expression is correlated with cardiovascular events. We studied RUNX1 binding to the PCTP promoter using DNA-protein binding studies and human erythroleukemia cells and promoter activity using luciferase reporter studies. We assessed the relationship between RUNX1 and PCTP in peripheral blood RNA and PCTP and death or myocardial infarction in 2 separate patient cohorts (587 total patients) with cardiovascular disease. Platelet PCTP protein in the patient was reduced by ≈50%. DNA-protein binding studies showed RUNX1 binding to consensus sites in ≈1 kB of PCTP promoter. PCTP expression was increased with RUNX1 overexpression and reduced with RUNX1 knockdown in human erythroleukemia cells, indicating that PCTP is regulated by RUNX1. Studies in 2 cohorts of patients showed that RUNX1 expression in blood correlated with PCTP gene expression; PCTP expression was higher in black compared with white subjects and was associated with future death/myocardial infarction after adjustment for age, sex, and race (odds ratio, 2.05; 95% confidence interval 1.6-2.7; P<0.0001). RUNX1 expression is known to initiate at 2 alternative promoters, a distal P1 and a proximal P2 promoter. In patient cohorts, there were differential effects of RUNX1

  10. ProDaMa: an open source Python library to generate protein structure datasets

    Directory of Open Access Journals (Sweden)

    Manconi Andrea

    2009-10-01

    Full Text Available Abstract Background The huge difference between the number of known sequences and known tertiary structures has justified the use of automated methods for protein analysis. Although a general methodology to solve these problems has not been yet devised, researchers are engaged in developing more accurate techniques and algorithms whose training plays a relevant role in determining their performance. From this perspective, particular importance is given to the training data used in experiments, and researchers are often engaged in the generation of specialized datasets that meet their requirements. Findings To facilitate the task of generating specialized datasets we devised and implemented ProDaMa, an open source Python library than provides classes for retrieving, organizing, updating, analyzing, and filtering protein data. Conclusion ProDaMa has been used to generate specialized datasets useful for secondary structure prediction and to develop a collaborative web application aimed at generating and sharing protein structure datasets. The library, the related database, and the documentation are freely available at the URL http://iasc.diee.unica.it/prodama.

  11. Gammaherpesvirus gene expression and DNA synthesis are facilitated by viral protein kinase and histone variant H2AX.

    Science.gov (United States)

    Mounce, Bryan C; Tsan, Fei Chin; Droit, Lindsay; Kohler, Sarah; Reitsma, Justin M; Cirillo, Lisa A; Tarakanova, Vera L

    2011-11-25

    Gammaherpesvirus protein kinases are an attractive therapeutic target as they support lytic replication and latency. Via an unknown mechanism these kinases enhance expression of select viral genes and DNA synthesis. Importantly, the kinase phenotypes have not been examined in primary cell types. Mouse gammaherpesvirus-68 (MHV68) protein kinase orf36 activates the DNA damage response (DDR) and facilitates lytic replication in primary macrophages. Significantly, H2AX, a DDR component and putative orf36 substrate, enhances MHV68 replication. Here we report that orf36 facilitated expression of RTA, an immediate early MHV68 gene, and DNA synthesis during de novo infection of primary macrophages. H2AX expression supported efficient RTA transcription and phosphorylated H2AX associated with RTA promoter. Furthermore, viral DNA synthesis was attenuated in H2AX-deficient macrophages, suggesting that the DDR system was exploited throughout the replication cycle. The interactions between a cancer-associated gammaherpesvirus and host tumor suppressor system have important implications for the pathogenesis of gammaherpesvirus infection.

  12. Generating a detailed protein profile of Fasciola hepatica during the chronic stage of infection in cattle.

    Science.gov (United States)

    Haçarız, Orçun; Baykal, Ahmet Tarık; Akgün, Mete; Kavak, Pınar; Sağıroğlu, Mahmut Şamil; Sayers, Gearóid Patrick

    2014-06-01

    Fasciola hepatica is a trematode helminth causing a damaging disease, fasciolosis, in ruminants and humans. Comprehensive proteomic studies broaden our knowledge of the parasite's protein profile, and provide new insights into the development of more effective strategies to deal with fasciolosis. The objective of this study was to generate a comprehensive profile of F. hepatica proteins expressed during the chronic stage of infection in cattle by building on previous efforts in this area. The approach included an improved sample preparation procedure for surface and internal layers of the parasite, the application of nano-UPLC-ESI-qTOF-MS (nano-ultra-performance LC and ESI quadrupole TOF MS) integrated with different acquisition methods and in silico database search against various protein databases and a transcript database including a new assembly of publically available EST. Of a total of 776 identified proteins, 206 and 332 were specific to the surface and internal layers of the parasite, respectively. Furthermore, 238 proteins were common to both layers, with comparative differences of 172 proteins detected. Specific proteins not previously identified in F. hepatica, but shown to be immunomodulatory or potential drug targets for other parasites, are discussed.

  13. Proteins at interfaces probed by chiral vibrational sum frequency generation spectroscopy.

    Science.gov (United States)

    Yan, Elsa C Y; Wang, Zhuguang; Fu, Li

    2015-02-19

    Characterizations of protein structures at interfaces are important in solving an array of fundamental and engineering problems, including understanding transmembrane signal transduction and molecular transport processes and development of biomaterials to meet the needs of biomedical and energy research. However, in situ and real-time characterization of protein secondary structures is challenging because it requires physical methods that are selective to both interface and secondary structures. Here, we summarize recent experimental developments in our laboratory of chiral vibrational sum frequency generation spectroscopy (SFG) for analyzing protein structures at interfaces. We showed that chiral SFG provides vibrational optical signatures of the peptide N-H stretch and amide I modes that can distinguish various protein secondary structures. Using these signatures, we further applied chiral SFG to probe orientations and folding kinetics of proteins at interfaces. Our results show that chiral SFG is a background-free, label-free, in situ, and real-time vibrational method for studying proteins at interfaces. This recent progress demonstrates the potential of chiral SFG in solving problems related to proteins and other chiral biopolymers at interfaces.

  14. Lewy Body Variant of Alzheimer's Disease: Selective Neocortical Loss of t-SNARE Proteins and Loss of MAP2 and α-Synuclein in Medial Temporal Lobe

    Directory of Open Access Journals (Sweden)

    Elizabeta B. Mukaetova-Ladinska

    2009-01-01

    Full Text Available Lewy bodies (LBs appear in the brains of nondemented individuals and also occur in a range of neurodegenerative disorders, such as dementia with Lewy bodies (DLB and Parkinson's disease. A number of people with a definite diagnosis of Alzheimer's disease (AD also exhibit these intraneuronal inclusions in allo- and/or neocortical areas. The latter, referred to as Lewy body variant of AD (LBV, bears a clinical resemblance to AD in terms of age at onset, duration of illness, cognitive impairment, and illness severity. Since the presence of LBs is accompanied by neuronal cytoskeleton changes, it is possible that the latter may influence neuronal connectivity via alterations to the synaptic network. To address this, we examined the expression of synaptic proteins (synaptophysin, syntaxin, SNAP-25, and α-synuclein and two cytoskeletal proteins (tau and MAP2 in the brain tissue of subjects enrolled in a population-based autopsy study (n = 47. They were divided into groups with no memory problems (control group, n = 15, LBV (n = 5, AD devoid of LBs (n = 17, cerebrovascular dementia (n = 3, and mixed dementia (n = 7. The LBV and AD groups had a similar degree of cognitive impairment and neuropathological staging in terms of Braak staging and CERAD score. In comparison with the control group and the dementia groups without LBs, the LBV group had significantly lower levels of syntaxin and SNAP-25 (23% in the neocortex, and depletion of MAP2 (64%, SNAP-25 (34%, and α-synuclein (44% proteins in the medial temporal lobes. These findings suggest that the t-SNARE complex deficit present in LBV may be associated with the presence of LB-related pathology and may explain the more profound cholinergic loss seen in these patients.

  15. A δ38 deletion variant of human transketolase as a model of transketolase-like protein 1 exhibits no enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Stefan Schneider

    Full Text Available Besides transketolase (TKT, a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1 has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a "pseudo-TKTL1" Δ38 deletion variant of human TKT (TKTΔ38 as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism.

  16. Fusion protein vaccines targeting two tumor antigens generate synergistic anti-tumor effects.

    Directory of Open Access Journals (Sweden)

    Wen-Fang Cheng

    Full Text Available INTRODUCTION: Human papillomavirus (HPV has been consistently implicated in causing several kinds of malignancies, and two HPV oncogenes, E6 and E7, represent two potential target antigens for cancer vaccines. We developed two fusion protein vaccines, PE(ΔIII/E6 and PE(ΔIII/E7 by targeting these two tumor antigens to test whether a combination of two fusion proteins can generate more potent anti-tumor effects than a single fusion protein. MATERIALS AND METHODS: In vivo antitumor effects including preventive, therapeutic, and antibody depletion experiments were performed. In vitro assays including intracellular cytokine staining and ELISA for Ab responses were also performed. RESULTS: PE(ΔIII/E6+PE(ΔIII/E7 generated both stronger E6 and E7-specific immunity. Only 60% of the tumor protective effect was observed in the PE(ΔIII/E6 group compared to 100% in the PE(ΔIII/E7 and PE(ΔIII/E6+PE(ΔIII/E7 groups. Mice vaccinated with the PE(ΔIII/E6+PE(ΔIII/E7 fusion proteins had a smaller subcutaneous tumor size than those vaccinated with PE(ΔIII/E6 or PE(ΔIII/E7 fusion proteins alone. CONCLUSION: Fusion protein vaccines targeting both E6 and E7 tumor antigens generated more potent immunotherapeutic effects than E6 or E7 tumor antigens alone. This novel strategy of targeting two tumor antigens together can promote the development of cancer vaccines and immunotherapy in HPV-related malignancies.

  17. Fusion Protein Vaccines Targeting Two Tumor Antigens Generate Synergistic Anti-Tumor Effects

    Science.gov (United States)

    Cheng, Wen-Fang; Chang, Ming-Cheng; Sun, Wei-Zen; Jen, Yu-Wei; Liao, Chao-Wei; Chen, Yun-Yuan; Chen, Chi-An

    2013-01-01

    Introduction Human papillomavirus (HPV) has been consistently implicated in causing several kinds of malignancies, and two HPV oncogenes, E6 and E7, represent two potential target antigens for cancer vaccines. We developed two fusion protein vaccines, PE(ΔIII)/E6 and PE(ΔIII)/E7 by targeting these two tumor antigens to test whether a combination of two fusion proteins can generate more potent anti-tumor effects than a single fusion protein. Materials and Methods In vivo antitumor effects including preventive, therapeutic, and antibody depletion experiments were performed. In vitro assays including intracellular cytokine staining and ELISA for Ab responses were also performed. Results PE(ΔIII)/E6+PE(ΔIII)/E7 generated both stronger E6 and E7-specific immunity. Only 60% of the tumor protective effect was observed in the PE(ΔIII)/E6 group compared to 100% in the PE(ΔIII)/E7 and PE(ΔIII)/E6+PE(ΔIII)/E7 groups. Mice vaccinated with the PE(ΔIII)/E6+PE(ΔIII)/E7 fusion proteins had a smaller subcutaneous tumor size than those vaccinated with PE(ΔIII)/E6 or PE(ΔIII)/E7 fusion proteins alone. Conclusion Fusion protein vaccines targeting both E6 and E7 tumor antigens generated more potent immunotherapeutic effects than E6 or E7 tumor antigens alone. This novel strategy of targeting two tumor antigens together can promote the development of cancer vaccines and immunotherapy in HPV-related malignancies. PMID:24058440

  18. Common genetic variants in fatty acid-binding protein-4 (FABP4) and clinical diabetes risk in the Women's Health Initiative Observational Study.

    Science.gov (United States)

    Chan, Kei-Hang K; Song, Yiqing; Hsu, Yi-Hsiang; You, Nai-Chieh Y; F Tinker, Lesley; Liu, Simin

    2010-09-01

    Adipocypte fatty acid-binding protein-4 (FABP4/adipocyte P2) may play a central role in energy metabolism and inflammation. In animal models, defects of the aP2 gene (aP2(-/-)) partially protected against the development of obesity-related insulin resistance, dyslipidemia, and atherosclerosis. However, it is unclear whether common genetic variation in FABP4 gene contributes to risk of type 2 diabetes (T2D) or diabetes-related metabolic traits in humans. We comprehensively assess the genetic associations of variants in the FABP4 gene with T2D risk and diabetes-associated biomarkers in a prospective study of 1,529 cases and 2,147 controls among postmenopausal women aged 50-79 years who enrolled in the Women's Health Initiative Observational Study (WHI-OS). We selected and genotyped a total of 11 haplotype-tagging single-nucleotide polymorphisms (tSNPs) spanning 41.3 kb across FABP4 in all samples. None of the SNPs and their derived haplotypes showed significant association with T2D risk. There were no significant associations between SNPs and plasma levels of inflammatory and endothelial biomarkers, including C-reactive protein, tumor necrosis factor (TNF), interleukin-6 (IL-6), E-selectin, and intercellular adhesion molecule (ICAM-1). Among African-American women, several SNPs were significantly associated with lower levels of vascular cell adhesion molecule-1 (VCAM-1), especially among those with incident T2D. On average, plasma levels of VCAM-1 were significantly lower among carriers of each minor allele at rs1486004(C/T; -1.08 ng/ml, P = 0.01), rs7017115(A/G; -1.07 ng/ml, P = 0.02), and rs2290201(C/T; -1.12 ng/ml, P = 0.002) as compared with the homozygotes of the common allele, respectively. After adjusting for multiple testing, carriers of the rs2290201 minor allele remained significantly associated with decreasing levels of plasma VCAM-1 in these women (P = 0.02). In conclusion, our finding from a multiethnic cohort of postmenopausal women did not support the

  19. Engineering Specificity from Broad to Narrow: Design of a β-Lactamase Inhibitory Protein (BLIP) Variant That Exclusively Binds and Detects KPC β-Lactamase.

    Science.gov (United States)

    Chow, Dar-Chone; Rice, Kacie; Huang, Wanzhi; Atmar, Robert L; Palzkill, Timothy

    2016-12-09

    The β-lactamase inhibitory protein (BLIP) binds and inhibits a wide range of class A β-lactamases including the TEM-1 β-lactamase (Ki = 0.5 nM), which is widely present in Gram-negative bacteria, and the KPC-2 β-lactamase (Ki = 1.2 nM), which hydrolyzes virtually all clinically useful β-lactam antibiotics. The extent to which the specificity of a protein that binds a broad range of targets can be modified to display narrow specificity was explored in this study by engineering BLIP to bind selectively to KPC-2 β-lactamase. A genetic screen for BLIP function in Escherichia coli was used to narrow the binding specificity of BLIP by identifying amino acid substitutions that retain affinity for KPC-2 while losing affinity for TEM-1 β-lactamase. The combination of single substitutions yielded the K74T:W112D BLIP variant, which was shown by inhibition assays to retain high affinity for KPC-2 with a Ki of 0.4 nM, while drastically losing affinity for TEM-1 with a Ki > 10 μM. The K74T:W112D mutant therefore binds KPC-2 β-lactamase 3 times more tightly while binding TEM-1 > 20000-fold more weakly than wild-type BLIP. The K74T:W112D BLIP variant also exhibited low affinity (Ki > 10 μM) for other class A β-lactamases. The high affinity and narrow specificity of BLIP K74T:W112D for KPC-2 β-lactamase suggest it could be a useful sensor for the presence of this enzyme in multidrug-resistant bacteria. This was demonstrated with an assay employing BLIP K74T:W112D conjugated to a bead to specifically pull-down and detect KPC-2 β-lactamase in lysates from clinical bacterial isolates containing multiple β-lactamases.

  20. Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide and protein peroxides generated by singlet oxygen attack

    DEFF Research Database (Denmark)

    Morgan, Philip E; Dean, Roger T; Davies, Michael Jonathan

    2002-01-01

    the active-site thiol of the enzyme and the peroxide. A number of low-molecular-mass compounds including thiols and ascorbate, but not Trolox C, can prevent inhibition by removing the initial peroxide, or species derived from it. In contrast, glutathione reductase and lactate dehydrogenase are poorly......Reaction of certain peptides and proteins with singlet oxygen (generated by visible light in the presence of rose bengal dye) yields long-lived peptide and protein peroxides. Incubation of these peroxides with glyceraldehyde-3-phosphate dehydrogenase, in the absence of added metal ions, results...

  1. A low-affinity penicillin-binding protein 2x variant is required for heteroresistance in Streptococcus pneumoniae.

    Science.gov (United States)

    Engel, Hansjürg; Mika, Moana; Denapaite, Dalia; Hakenbeck, Regine; Mühlemann, Kathrin; Heller, Manfred; Hathaway, Lucy J; Hilty, Markus

    2014-07-01

    Heteroresistance to penicillin in Streptococcus pneumoniae is the ability of subpopulations to grow at a higher antibiotic concentration than expected from the MIC. This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin-binding protein 2b (PBP2b) and PBP2x, and the secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from the heteroresistant strain Spain(23F) 2349 in the nonheteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found that pbp2x, but not pbp2b or pbp1a alone, conferred heteroresistance to R6. However, a change of pbp2x expression was not observed, and therefore, expression does not correlate with an increased proportion of resistant subpopulations. In addition, the influence of the CiaRH system, mediating PBP-independent β-lactam resistance, was assessed by PAP on ciaR disruption mutants but revealed no heteroresistant phenotype. We also showed that the highly resistant subpopulations (HOM*) of transformants containing low-affinity pbp2x undergo an increase in resistance upon selection on penicillin plates that partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins encoded by pstS, phoU, pstB, and pstC in these highly resistant subpopulations. In conclusion, the presence of low-affinity pbp2x enables certain pneumococcal colonies to survive in the presence of β-lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaptation to antibiotic stress.

  2. Distinct composition of bovine milk from Jersey and Holstein-Friesian cows with good, poor, or noncoagulation properties as reflected in protein genetic variants and isoforms.

    Science.gov (United States)

    Jensen, H B; Poulsen, N A; Andersen, K K; Hammershøj, M; Poulsen, H D; Larsen, L B

    2012-12-01

    The objective of this study was to examine variation in overall milk, protein, and mineral composition of bovine milk in relation to rennet-induced coagulation, with the aim of elucidating the underlying causes of milk with impaired coagulation abilities. On the basis of an initial screening of 892 milk samples from 42 herds with Danish Jersey and Holstein-Friesian cows, a subset of 102 samples was selected to represent milk with good, poor, or noncoagulating properties (i.e., samples that within each breed represented the most extremes in regard to coagulation properties). Milk with good coagulation characteristics was defined as milk forming a strong coagulum based on oscillatory rheology, as indicated by high values for maximum coagulum strength (G'(max)) and curd firming rate (CFR) and a short rennet coagulation time. Poorly coagulating milk formed a weak coagulum, with a low G'(max) and CFR and a long rennet coagulation time. Noncoagulating milk was defined as milk that failed to form a coagulum, having G'(max) and CFR values of zero at measurements taken within 1h after addition of rennet. For both breeds, a lower content of total protein, total casein (CN) and κ-CN, and lower levels of minerals (Ca, P, Mg) were identified in poorly coagulating and noncoagulating milk in comparison with milk with good coagulation properties. Liquid chromatography/electrospray ionization-mass spectrometry revealed the presence of a great variety of genetic variants of the major milk proteins, namely, α(S1)-CN (variants B and C), α(S2)-CN (A), β-CN (A(1), A(2), B, I, and F), κ-CN (A, B, and E), α-lactalbumin (B), and β-lactoglobulin (A, B, and C). In poorly coagulating and noncoagulating milk samples of both breeds, the predominant composite genotype of α(S1)-, β-, and κ-CN was BB-A(2)A(2)-AA, which confirmed a genetic contribution to impaired milk coagulation. Interestingly, subtle variations in posttranslational modification of CN were observed between the

  3. Alternative polyadenylation in a family of paralogous EPB41 genes generates protein 4.1 diversity.

    Science.gov (United States)

    Rangel, Laura; Lospitao, Eva; Ruiz-Sáenz, Ana; Alonso, Miguel A; Correas, Isabel

    2017-02-01

    Alternative polyadenylation (APA) is a step in mRNA 3'-end processing that contributes to the complexity of the transcriptome by generating isoforms that differ in either their coding sequence or their 3'-untranslated regions (UTRs). The EPB41 genes, EPB41, EPB41L2, EPB41L3 and EPB41L1, encode an impressively complex array of structural adaptor proteins (designated 4.1R, 4.1G, 4.1B and 4.1N, respectively) by using alternative transcriptional promoters and tissue-specific alternative pre-mRNA splicing. The great variety of 4.1 proteins mainly results from 5'-end and internal processing of the EPB41 pre-mRNAs. Thus, 4.1 proteins can vary in their N-terminal extensions but all contain a highly homologous C-terminal domain (CTD). Here we study a new group of EPB41-related mRNAs that originate by APA and lack the exons encoding the CTD characteristic of prototypical 4.1 proteins, thereby encoding a new type of 4.1 protein. For the EPB41 gene, this type of processing was observed in all 11 human tissues analyzed. Comparative genomic analysis of EPB41 indicates that APA is conserved in various mammals. In addition, we show that APA also functions for the EPB41L2, EPB41L3 and EPB41L1 genes, but in a more restricted manner in the case of the latter 2 than it does for the EPB41 and EPB41L2 genes. Our study shows alternative polyadenylation to be an additional mechanism for the generation of 4.1 protein diversity in the already complex EPB41-related genes. Understanding the diversity of EPB41 RNA processing is essential for a full appreciation of the many 4.1 proteins expressed in normal and pathological tissues.

  4. Automatic generation of 3D motifs for classification of protein binding sites

    Directory of Open Access Journals (Sweden)

    Herzyk Pawel

    2007-08-01

    Full Text Available Abstract Background Since many of the new protein structures delivered by high-throughput processes do not have any known function, there is a need for structure-based prediction of protein function. Protein 3D structures can be clustered according to their fold or secondary structures to produce classes of some functional significance. A recent alternative has been to detect specific 3D motifs which are often associated to active sites. Unfortunately, there are very few known 3D motifs, which are usually the result of a manual process, compared to the number of sequential motifs already known. In this paper, we report a method to automatically generate 3D motifs of protein structure binding sites based on consensus atom positions and evaluate it on a set of adenine based ligands. Results Our new approach was validated by generating automatically 3D patterns for the main adenine based ligands, i.e. AMP, ADP and ATP. Out of the 18 detected patterns, only one, the ADP4 pattern, is not associated with well defined structural patterns. Moreover, most of the patterns could be classified as binding site 3D motifs. Literature research revealed that the ADP4 pattern actually corresponds to structural features which show complex evolutionary links between ligases and transferases. Therefore, all of the generated patterns prove to be meaningful. Each pattern was used to query all PDB proteins which bind either purine based or guanine based ligands, in order to evaluate the classification and annotation properties of the pattern. Overall, our 3D patterns matched 31% of proteins with adenine based ligands and 95.5% of them were classified correctly. Conclusion A new metric has been introduced allowing the classification of proteins according to the similarity of atomic environment of binding sites, and a methodology has been developed to automatically produce 3D patterns from that classification. A study of proteins binding adenine based ligands showed that

  5. Thermodynamics and mechanics of membrane curvature generation and sensing by proteins and lipids.

    Science.gov (United States)

    Baumgart, Tobias; Capraro, Benjamin R; Zhu, Chen; Das, Sovan L

    2011-01-01

    Research investigating lipid membrane curvature generation and sensing is a rapidly developing frontier in membrane physical chemistry and biophysics. The fast recent progress is based on the discovery of a plethora of proteins involved in coupling membrane shape to cellular membrane function, the design of new quantitative experimental techniques to study aspects of membrane curvature, and the development of analytical theories and simulation techniques that allow a mechanistic interpretation of quantitative measurements. The present review first provides an overview of important classes of membrane proteins for which function is coupled to membrane curvature. We then survey several mechanisms that are assumed to underlie membrane curvature sensing and generation. Finally, we discuss relatively simple thermodynamic/mechanical models that allow quantitative interpretation of experimental observations.

  6. Genetic Variants of GPER/GPR30, a Novel Estrogen-Related G Protein Receptor, Are Associated with Human Seminoma

    Science.gov (United States)

    Chevalier, Nicolas; Paul-Bellon, Rachel; Camparo, Philippe; Michiels, Jean-François; Chevallier, Daniel; Fénichel, Patrick

    2014-01-01

    Testicular germ cell tumors (TGCTs) are the most common solid cancers in young men, with an increasing incidence over several years. However, their pathogenesis remains a matter of debate. Some epidemiological data suggest the involvement of both environmental and genetic factors. We reported two distinct effects of estrogens and/or xeno-estrogens on in vitro human seminoma-derived cells proliferation: (1) an antiproliferative effect via a classical estrogen receptor beta-dependent pathway, and (2) a promotive effect via a non-classical membrane G-protein-coupled receptor, GPR30/GPER, which is only overexpressed in seminomas, the most common TGCT. In order to explain this overexpression, we investigated the possible association of polymorphisms in the GPER gene by using allele-specific tetra-primer polymerase chain reaction performed on tissue samples from 150 paraffin-embedded TGCT specimens (131 seminomas, 19 non seminomas). Compared to control population, loss of homozygous ancestral genotype GG in two polymorphisms located in the promoter region of GPER (rs3808350 and rs3808351) was more frequent in seminomas but not in non-seminomas (respectively, OR = 1.960 (1.172–3.277) and 7.000 (2.747–17.840); p < 0.01). These polymorphisms may explain GPER overexpression and represent a genetic factor of susceptibility supporting the contribution of environmental GPER ligands in testicular carcinogenesis. PMID:24451139

  7. Genetic Variants of GPER/GPR30, a Novel Estrogen-Related G Protein Receptor, Are Associated with Human Seminoma

    Directory of Open Access Journals (Sweden)

    Nicolas Chevalier

    2014-01-01

    Full Text Available Testicular germ cell tumors (TGCTs are the most common solid cancers in young men, with an increasing incidence over several years. However, their pathogenesis remains a matter of debate. Some epidemiological data suggest the involvement of both environmental and genetic factors. We reported two distinct effects of estrogens and/or xeno-estrogens on in vitro human seminoma-derived cells proliferation: (1 an antiproliferative effect via a classical estrogen receptor beta-dependent pathway, and (2 a promotive effect via a non-classical membrane G-protein-coupled receptor, GPR30/GPER, which is only overexpressed in seminomas, the most common TGCT. In order to explain this overexpression, we investigated the possible association of polymorphisms in the GPER gene by using allele-specific tetra-primer polymerase chain reaction performed on tissue samples from 150 paraffin-embedded TGCT specimens (131 seminomas, 19 non seminomas. Compared to control population, loss of homozygous ancestral genotype GG in two polymorphisms located in the promoter region of GPER (rs3808350 and rs3808351 was more frequent in seminomas but not in non-seminomas (respectively, OR = 1.960 (1.172–3.277 and 7.000 (2.747–17.840; p < 0.01. These polymorphisms may explain GPER overexpression and represent a genetic factor of susceptibility supporting the contribution of environmental GPER ligands in testicular carcinogenesis.

  8. Generation and characterization of a monoclonal antibody against prM protein of West Nile virus.

    Science.gov (United States)

    Guo, Li-Ping; Huo, Hong; Wang, Xiao-Lei; Bu, Zhi-Gao; Hua, Rong-Hong

    2014-12-01

    West Nile virus (WNV), which is an emerging pathogenic flavivirus with increasing distribution worldwide, is the cause of major human and animal health concerns. The pre-membrane (prM) protein of WNV is cleaved during maturation by the furin protease into the structural protein M and a pr-segment. In this study we generated and characterized a monoclonal antibody (MAb) against the WNV prM protein. Western blot analysis showed that the MAb reacted with WNV prM specifically. Immunohistochemistry assays demonstrated that the MAb recognized native prM protein in transfected BHK-21 cells. Preliminary studies were performed to identify the epitope recognized by the MAb using a set of synthesized overlapping peptides spanning the whole length of the prM protein. The MAb reported here may provide a valuable tool for the further exploration of the biological properties and functions of the prM protein and may also be developed for potential clinical applications.

  9. Structure and orientation of interfacial proteins determined by sum frequency generation vibrational spectroscopy: method and application.

    Science.gov (United States)

    Ye, Shuji; Wei, Feng; Li, Hongchun; Tian, Kangzhen; Luo, Yi

    2013-01-01

    In situ and real-time characterization of molecular structures and orientation of proteins at interfaces is essential to understand the nature of interfacial protein interaction. Such work will undoubtedly provide important clues to control biointerface in a desired manner. Sum frequency generation vibrational spectroscopy (SFG-VS) has been demonstrated to be a powerful technique to study the interfacial structures and interactions at the molecular level. This paper first systematically introduced the methods for the calculation of the Raman polarizability tensor, infrared transition dipole moment, and SFG molecular hyperpolarizability tensor elements of proteins/peptides with the secondary structures of α-helix, 310-helix, antiparallel β-sheet, and parallel β-sheet, as well as the methodology to determine the orientation of interfacial protein secondary structures using SFG amide I spectra. After that, recent progresses on the determination of protein structure and orientation at different interfaces by SFG-VS were then reviewed, which provides a molecular-level understanding of the structures and interactions of interfacial proteins, specially understanding the nature of driving force behind such interactions. Although this review has focused on analysis of amide I spectra, it will be expected to offer a basic idea for the spectral analysis of amide III SFG signals and other complicated molecular systems such as RNA and DNA. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Human variant Creutzfeldt-Jakob disease and sheep scrapie PrP(res) detection using seeded conversion of recombinant prion protein.

    Science.gov (United States)

    Orrú, Christina D; Wilham, Jason M; Hughson, Andrew G; Raymond, Lynne D; McNally, Kristin L; Bossers, Alex; Ligios, Ciriaco; Caughey, Byron

    2009-08-01

    The pathological isoform of the prion protein (PrP(res)) can serve as a marker for prion diseases, but more practical tests are needed for preclinical diagnosis and sensitive detection of many prion infections. Previously we showed that the quaking-induced conversion (QuIC) assay can detect sub-femtogram levels of PrP(res) in scrapie-infected hamster brain tissue and distinguish cerebral spinal fluid (CSF) samples from normal and scrapie-infected hamsters. We now report the adaptation of the QuIC reaction to prion diseases of medical and agricultural interest: human variant Creutzfeldt-Jakob disease (vCJD) and sheep scrapie. PrP(res)-positive and -negative brain homogenates from humans and sheep were discriminated within 1-2 days with a sensitivity of 10-100 fg PrP(res). More importantly, in as little as 22 h we were able to distinguish CSF samples from scrapie-infected and uninfected sheep. These results suggest the presence of prions in CSF from scrapie-infected sheep. This new method enables the relatively rapid and sensitive detection of human CJD and sheep scrapie PrP(res) and may facilitate the development of practical preclinical diagnostic and high-throughput interference tests.

  11. Pinkbar is an epithelial-specific BAR domain protein that generates planar membrane structures

    Energy Technology Data Exchange (ETDEWEB)

    Pykäläinen, Anette; Boczkowska, Malgorzata; Zhao, Hongxia; Saarikangas, Juha; Rebowski, Grzegorz; Jansen, Maurice; Hakanen, Janne; Koskela, Essi V.; Peränen, Johan; Vihinen, Helena; Jokitalo, Eija; Salminen, Marjo; Ikonen, Elina; Dominguez, Roberto; Lappalainen, Pekka (Helsinki); (Penn)

    2013-05-29

    Bin/amphipysin/Rvs (BAR)-domain proteins sculpt cellular membranes and have key roles in processes such as endocytosis, cell motility and morphogenesis. BAR domains are divided into three subfamilies: BAR- and F-BAR-domain proteins generate positive membrane curvature and stabilize cellular invaginations, whereas I-BAR-domain proteins induce negative curvature and stabilize protrusions. We show that a previously uncharacterized member of the I-BAR subfamily, Pinkbar, is specifically expressed in intestinal epithelial cells, where it localizes to Rab13-positive vesicles and to the plasma membrane at intercellular junctions. Notably, the BAR domain of Pinkbar does not induce membrane tubulation but promotes the formation of planar membrane sheets. Structural and mutagenesis analyses reveal that the BAR domain of Pinkbar has a relatively flat lipid-binding interface and that it assembles into sheet-like oligomers in crystals and in solution, which may explain its unique membrane-deforming activity.

  12. Large-scale analysis of association between LRP5 and LRP6 variants and osteoporosis

    NARCIS (Netherlands)

    J.B.J. van Meurs (Joyce); T.A. Trikalinos (Thomas); S.H. Ralston (Stuart); S. Balcells (Susana); M.L. Brandi; K. Brixen (Kim); D.P. Kiel (Douglas); B.L. Langdahl (Bente); P. Lips (Paul); O. Ljunggren (Östen); R. Lorenc (Roman); B. Obermayer-Pietsch (Barbara); C. Ohlsson (Claes); U. Pettersson (Ulrika); D.M. Reid (David); M.F. Rousseau (Francois); S. Scollen (Serena); W. Van Hul (Wim); L. Agueda (Lidia); K. Åkesson (Kristina); L.I. Benevolenskaya (Lidia); S.L. Ferrari (Serge); G. Hallmans (Göran); A. Hofman (Albert); L.B. Husted (Lise Bjerre); M. Kruk (Marcin); S. Kaptoge (Stephen); D. Karasik (David); M. Karlsson (Magnus); M. Lorentzon (Mattias); L. Masi (Laura); F.E. McGuigan; D. Mellström (Dan); L. Mosekilde (Leif); X. Nogues (Xavier); H.A.P. Pols (Huib); J. Reeve (Jonathan); W. Renner (Wilfried); F. Rivadeneira Ramirez (Fernando); N.M. van Schoor (Natasja); K. Weber (Kurt); J.P.A. Ioannidis (John); A.G. Uitterlinden (André)

    2008-01-01

    textabstractContext: Mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene cause rare syndromes characterized by altered bone mineral density (BMD). More common LRP5 variants may affect osteoporosis risk in the general population. Objective: To generate large-scale evidence

  13. Large-scale analysis of association between LRP5 and LRP6 variants and osteoporosis

    NARCIS (Netherlands)

    J.B.J. van Meurs (Joyce); T.A. Trikalinos (Thomas); S.H. Ralston (Stuart); S. Balcells (Susana); M.L. Brandi; K. Brixen (Kim); D.P. Kiel (Douglas); B.L. Langdahl (Bente); P. Lips (Paul); O. Ljunggren (Östen); R. Lorenc (Roman); B. Obermayer-Pietsch (Barbara); C. Ohlsson (Claes); U. Pettersson (Ulrika); D.M. Reid (David); M.F. Rousseau (Francois); S. Scollen (Serena); W. Van Hul (Wim); L. Agueda (Lidia); K. Åkesson (Kristina); L.I. Benevolenskaya (Lidia); S.L. Ferrari (Serge); G. Hallmans (Göran); A. Hofman (Albert); L.B. Husted (Lise Bjerre); M. Kruk (Marcin); S. Kaptoge (Stephen); D. Karasik (David); M. Karlsson (Magnus); M. Lorentzon (Mattias); L. Masi (Laura); F.E. McGuigan; D. Mellström (Dan); L. Mosekilde (Leif); X. Nogues (Xavier); H.A.P. Pols (Huib); J. Reeve (Jonathan); W. Renner (Wilfried); F. Rivadeneira Ramirez (Fernando); N.M. van Schoor (Natasja); K. Weber (Kurt); J.P.A. Ioannidis (John); A.G. Uitterlinden (André)

    2008-01-01

    textabstractContext: Mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene cause rare syndromes characterized by altered bone mineral density (BMD). More common LRP5 variants may affect osteoporosis risk in the general population. Objective: To generate large-scale evidence

  14. Linoleic acid-induced mitochondrial Ca(2+) efflux causes peroxynitrite generation and protein nitrotyrosylation.

    Science.gov (United States)

    Zhang, Hong-Mei; Dang, Howard; Yeh, Chih-Ko; Zhang, Bin-Xian

    2009-06-26

    It is well known that excessive non-esterified fatty acids in diabetes contribute to the pathogenesis of renal complications although the mechanism remains elusive. Enhanced oxidative stress has been hypothesized as a unified factor contributing to diabetic complications and increased protein nitrotyrosylation has been reported in the kidneys of diabetic patients. In the current manuscript we described that linoleic acid (LA) caused mitochondrial Ca(2+) efflux and peroxynitrite production, along with increased nitrotyrosine levels of cellular proteins in primary human mesangial cells. The peroxynitrite production by LA was found to depend on mitochondrial Ca(2+) efflux. Downregulation of hsp90beta1, which has been previously shown to be essential for polyunsaturated fatty acid-induced mitochondrial Ca(2+) efflux, significantly diminished LA-responsive mitochondrial Ca(2+) efflux and the coupled peroxynitrite generation, implicating a critical role of hsp90beta1 in the LA responses. Our results further demonstrated that mitochondrial complexes I and III were directly involved in the LA-induced peroxynitrite generation. Using the well established type 2 diabetic animal model db/db mice, we observed a dramatically enhanced LA responsive mitochondrial Ca(2+) efflux and protein nitrotyrosylation in the kidney. Our study thus demonstrates a cause-effect relationship between LA and peroxynitrite or protein nitrotyrosylation and provides a novel mechanism for lipid-induced nephropathy in diabetes.

  15. Linoleic Acid-Induced Mitochondrial Ca2+ Efflux Causes Peroxynitrite Generation and Protein Nitrotyrosylation

    Science.gov (United States)

    Zhang, Hong-Mei; Dang, Howard; Yeh, Chih-Ko; Zhang, Bin-Xian

    2009-01-01

    It is well known that excessive non-esterified fatty acids in diabetes contribute to the pathogenesis of renal complications although the mechanism remains elusive. Enhanced oxidative stress has been hypothesized as a unified factor contributing to diabetic complications and increased protein nitrotyrosylation has been reported in the kidneys of diabetic patients. In the current manuscript we described that linoleic acid (LA) caused mitochondrial Ca2+ efflux and peroxynitrite production, along with increased nitrotyrosine levels of cellular proteins in primary human mesangial cells. The peroxynitrite production by LA was found to depend on mitochondrial Ca2+ efflux. Downregulation of hsp90β1, which has been previously shown to be essential for polyunsaturated fatty acid-induced mitochondrial Ca2+ efflux, significantly diminished LA-responsive mitochondrial Ca2+ efflux and the coupled peroxynitrite generation, implicating a critical role of hsp90β1 in the LA responses. Our results further demonstrated that mitochondrial complexes I and III were directly involved in the LA-induced peroxynitrite generation. Using the well established type 2 diabetic animal model db/db mice, we observed a dramatically enhanced LA responsive mitochondrial Ca2+ efflux and protein nitrotyrosylation in the kidney. Our study thus demonstrates a cause-effect relationship between LA and peroxynitrite or protein nitrotyrosylation and provides a novel mechanism for lipid-induced nephropathy in diabetes. PMID:19557129

  16. Linoleic acid-induced mitochondrial Ca(2+ efflux causes peroxynitrite generation and protein nitrotyrosylation.

    Directory of Open Access Journals (Sweden)

    Hong-Mei Zhang

    Full Text Available It is well known that excessive non-esterified fatty acids in diabetes contribute to the pathogenesis of renal complications although the mechanism remains elusive. Enhanced oxidative stress has been hypothesized as a unified factor contributing to diabetic complications and increased protein nitrotyrosylation has been reported in the kidneys of diabetic patients. In the current manuscript we described that linoleic acid (LA caused mitochondrial Ca(2+ efflux and peroxynitrite production, along with increased nitrotyrosine levels of cellular proteins in primary human mesangial cells. The peroxynitrite production by LA was found to depend on mitochondrial Ca(2+ efflux. Downregulation of hsp90beta1, which has been previously shown to be essential for polyunsaturated fatty acid-induced mitochondrial Ca(2+ efflux, significantly diminished LA-responsive mitochondrial Ca(2+ efflux and the coupled peroxynitrite generation, implicating a critical role of hsp90beta1 in the LA responses. Our results further demonstrated that mitochondrial complexes I and III were directly involved in the LA-induced peroxynitrite generation. Using the well established type 2 diabetic animal model db/db mice, we observed a dramatically enhanced LA responsive mitochondrial Ca(2+ efflux and protein nitrotyrosylation in the kidney. Our study thus demonstrates a cause-effect relationship between LA and peroxynitrite or protein nitrotyrosylation and provides a novel mechanism for lipid-induced nephropathy in diabetes.

  17. La-related protein 4 binds poly(A), interacts with the poly(A)-binding protein MLLE domain via a variant PAM2w motif, and can promote mRNA stability.

    Science.gov (United States)

    Yang, Ruiqing; Gaidamakov, Sergei A; Xie, Jingwei; Lee, Joowon; Martino, Luigi; Kozlov, Guennadi; Crawford, Amanda K; Russo, Amy N; Conte, Maria R; Gehring, Kalle; Maraia, Richard J

    2011-02-01

    The conserved RNA binding protein La recognizes UUU-3'OH on its small nuclear RNA ligands and stabilizes them against 3'-end-mediated decay. We report that newly described La-related protein 4 (LARP4) is a factor that can bind poly(A) RNA and interact with poly(A) binding protein (PABP). Yeast two-hybrid analysis and reciprocal immunoprecipitations (IPs) from HeLa cells revealed that LARP4 interacts with RACK1, a 40S ribosome- and mRNA-associated protein. LARP4 cosediments with 40S ribosome subunits and polyribosomes, and its knockdown decreases translation. Mutagenesis of the RNA binding or PABP interaction motifs decrease LARP4 association with polysomes. Several translation and mRNA metabolism-related proteins use a PAM2 sequence containing a critical invariant phenylalanine to make direct contact with the MLLE domain of PABP, and their competition for the MLLE is thought to regulate mRNA homeostasis. Unlike all ∼150 previously analyzed PAM2 sequences, LARP4 contains a variant PAM2 (PAM2w) with tryptophan in place of the phenylalanine. Binding and nuclear magnetic resonance (NMR) studies have shown that a peptide representing LARP4 PAM2w interacts with the MLLE of PABP within the affinity range measured for other PAM2 motif peptides. A cocrystal of PABC bound to LARP4 PAM2w shows tryptophan in the pocket in PABC-MLLE otherwise occupied by phenylalanine. We present evidence that LARP4 expression stimulates luciferase reporter activity by promoting mRNA stability, as shown by mRNA decay analysis of luciferase and cellular mRNAs. We propose that LARP4 activity is integrated with other PAM2 protein activities by PABP as part of mRNA homeostasis.

  18. The Duffy-Binding-Like β domain (DBLβ) of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variant, PFD1235w, binds ICAM1

    DEFF Research Database (Denmark)

    Andersen, M. A.; Bengtsson, A.; Rask, Thomas Salhøj

    2012-01-01

    Plasmodium falciparum is by far the most virulent human malaria parasite. P. falciparum variant erythrocyte surface antigens, known as PfEMP1, play a crucial role in malaria pathogenesis as they mediate adhesion to host endothelial receptors. The PfEMP1 variant, PFD1235w, encoded by the 3D7 group...

  19. Novel antigen design for the generation of antibodies to G-protein-coupled receptors.

    Science.gov (United States)

    Larsson, K; Hofström, C; Lindskog, C; Hansson, M; Angelidou, P; Hökfelt, T; Uhlén, M; Wernérus, H; Gräslund, T; Hober, S

    2011-07-29

    Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.

  20. Adjuvant effects elicited by novel oligosaccharide variants of detoxified meningococcal lipopolysaccharides on Neisseria meningitidis recombinant PorA protein: a comparison in mice.

    Directory of Open Access Journals (Sweden)

    Ojas H Mehta

    Full Text Available Neisseria meningitidis lipopolysaccharide (LPS has adjuvant properties that can be exploited to assist vaccine immunogenicity. The modified penta-acylated LPS retains the adjuvant properties of hexa-acylated LPS but has a reduced toxicity profile. In this study we investigated whether two modified glycoform structures (LgtE and IcsB of detoxified penta-acylated LPS exhibited differential adjuvant properties when formulated as native outer membrane vesicles (nOMVs as compared to the previously described LgtB variant. Detoxified penta-acylated LPS was obtained by disruption of the lpxL1 gene (LpxL1 LPS, and three different glycoforms were obtained by disruption of the lgtB, lgtE or icsB genes respectively. Mice (mus musculus were immunized with a recombinant PorA P1.7-2,4 (rPorA protein co-administered with different nOMVs (containing a different PorA serosubtype P1.7,16, each of which expressed one of the three penta-acylated LPS glycoforms. All nOMVs induced IgG responses against the rPorA, but the nOMVs containing the penta-acylated LgtB-LpxL1 LPS glycoform induced significantly greater bactericidal activity compared to the other nOMVs or when the adjuvant was Alhydrogel. Compared to LgtE or IcsB LPS glycoforms, these data support the use of nOMVs containing detoxified, modified LgtB-LpxL1 LPS as a potential adjuvant for future meningococcal protein vaccines.

  1. Lifestyle Intervention for Weight Loss and Cardiometabolic Changes in the Setting of Glucokinase Regulatory Protein (GCKR) Inhibition: GCKR-Leu446Pro Variant in Look AHEAD

    Science.gov (United States)

    Belalcazar, L. Maria; Papandonatos, George D.; Erar, Bahar; Peter, Inga; Alkofide, Hadeel; Balasubramanyam, Ashok; Brautbar, Ariel; Kahn, Steven E.; Knowler, William C.; Ballantyne, Christie M.; McCaffery, Jeanne M.; Huggins, Gordon S.

    2015-01-01

    Background Glucokinase regulatory protein (GCKR) inhibitors offer a novel treatment approach for glucose control in diabetes; however their cardiometabolic effects, particularly in relation to increased triglycerides and C-reactive protein (CRP) levels are of concern. GCKR Leu446Pro is a common variant associated with reduced GCKR function, increased triglycerides and CRP. Methods and Results We investigated whether a 1-year intensive lifestyle intervention for weight loss (ILI) would avert the unfavorable cardiometabolic effects associated with GCKR Leu446Pro when compared to a diabetes support and education arm (DSE) in overweight/obese individuals with type 2 diabetes with triglyceride (n=3,214) and CRP (n=1,411) data participating in a randomized lifestyle intervention study for weight loss, Look AHEAD (Action for Health in Diabetes). Once demographics, medication use and baseline adiposity and fitness were accounted for, ILI did not modify the baseline association of GCKR-Leu446Pro with elevated triglycerides (β± SE= 0.067 ± 0.013, p= 1.5×10−7 and β± SE= 0.052 ± 0.015, p=5×10−4) or with elevated CRP (β± SE= 0.136 ± 0.034, p=5.1×10−5and β± SE= 0.903 ± 0.038, p=0.015) in the overall sample and Non-Hispanic Whites, respectively. The lack of a protective effect from ILI at 1-year when compared to DSE (ILI versus DSE interaction for triglyceride and CRP change, respectively: p= 0.64 and 0.37 in the overall sample; p= 0.27 and 0.05 in Non-Hispanic Whites) persisted after additional adjustment for changes in adiposity and fitness. Conclusions Moderate improvements in adiposity and fitness with ILI did not mitigate the adverse cardiometabolic effects of GCKR inhibition in overweight/obese individuals with diabetes. PMID:26578543

  2. Rare, protein-truncating variants in ATM, CHEK2 and PALB2, but not XRCC2, are associated with increased breast cancer risks.

    Science.gov (United States)

    Decker, Brennan; Allen, Jamie; Luccarini, Craig; Pooley, Karen A; Shah, Mitul; Bolla, Manjeet K; Wang, Qin; Ahmed, Shahana; Baynes, Caroline; Conroy, Don M; Brown, Judith; Luben, Robert; Ostrander, Elaine A; Pharoah, Paul Dp; Dunning, Alison M; Easton, Douglas F

    2017-08-04

    Breast cancer (BC) is the most common malignancy in women and has a major heritable component. The risks associated with most rare susceptibility variants are not well estimated. To better characterise the contribution of variants in ATM, CHEK2, PALB2 and XRCC2, we sequenced their coding regions in 13 087 BC cases and 5488 controls from East Anglia, UK. Gene coding regions were enriched via PCR, sequenced, variant called and filtered for quality. ORs for BC risk were estimated separately for carriers of truncating variants and of rare missense variants, which were further subdivided by functional domain and pathogenicity as predicted by four in silico algorithms. Truncating variants in PALB2 (OR=4.69, 95% CI 2.27 to 9.68), ATM (OR=3.26; 95% CI 1.82 to 6.46) and CHEK2 (OR=3.11; 95% CI 2.15 to 4.69), but not XRCC2 (OR=0.94; 95% CI 0.26 to 4.19) were associated with increased BC risk. Truncating variants in ATM and CHEK2 were more strongly associated with risk of oestrogen receptor (ER)-positive than ER-negative disease, while those in PALB2 were associated with similar risks for both subtypes. There was also some evidence that missense variants in ATM, CHEK2 and PALB2 may contribute to BC risk, but larger studies are necessary to quantify the magnitude of this effect. Truncating variants in PALB2 are associated with a higher risk of BC than those in ATM or CHEK2. A substantial risk of BC due to truncating XRCC2 variants can be excluded. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  3. Evaluation of protective potential of Yersinia pestis outer membrane protein antigens as possible candidates for a new-generation recombinant plague vaccine.

    Science.gov (United States)

    Erova, Tatiana E; Rosenzweig, Jason A; Sha, Jian; Suarez, Giovanni; Sierra, Johanna C; Kirtley, Michelle L; van Lier, Christina J; Telepnev, Maxim V; Motin, Vladimir L; Chopra, Ashok K

    2013-02-01

    Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1(-) strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1(-) mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1(-) CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.

  4. Evaluation of Protective Potential of Yersinia pestis Outer Membrane Protein Antigens as Possible Candidates for a New-Generation Recombinant Plague Vaccine

    Science.gov (United States)

    Erova, Tatiana E.; Rosenzweig, Jason A.; Sha, Jian; Suarez, Giovanni; Sierra, Johanna C.; Kirtley, Michelle L.; van Lier, Christina J.; Telepnev, Maxim V.; Motin, Vladimir L.

    2013-01-01

    Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1− strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1− mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1− CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains. PMID:23239803

  5. Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic Escherichia coli

    Science.gov (United States)

    Luna-Pineda, Víctor M.; Reyes-Grajeda, Juan Pablo; Cruz-Córdova, Ariadnna; Saldaña-Ahuactzi, Zeus; Ochoa, Sara A.; Maldonado-Bernal, Carmen; Cázares-Domínguez, Vicenta; Moreno-Fierros, Leticia; Arellano-Galindo, José; Hernández-Castro, Rigoberto; Xicohtencatl-Cortes, Juan

    2016-01-01

    Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenic Escherichia coli (UPEC) is the main etiologic agent. Fimbriae assembled on the bacterial surface are essential for adhesion to the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules for use as potential target vaccines against UTIs. The fusion protein design was generated using bioinformatics tools, and template fusion gene sequences were synthesized by GenScript in the following order fimH-csgA-papG-fimH-csgA (fcpfc) linked to the nucleotide sequence encoding the [EAAAK]5 peptide. Monomeric (fimH, csgA, and papG), dimeric (fimH-csgA), and trimeric (fimH-csgA-papG) genes were cloned into the pLATE31 expression vector and generated products of 1040, 539, 1139, 1442, and 2444 bp, respectively. Fusion protein expression in BL21 E. coli was induced with 1 mM IPTG, and His-tagged proteins were purified under denaturing conditions and refolded by dialysis using C-buffer. Coomassie blue-stained SDS-PAGE gels and Western blot analysis revealed bands of 29.5, 11.9, 33.9, 44.9, and 82.1 kDa, corresponding to FimH, CsgA, PapG, FC, and FCP proteins, respectively. Mass spectrometry analysis by MALDI-TOF/TOF revealed specific peptides that confirmed the fusion protein structures. Dynamic light scattering analysis revealed the polydispersed state of the fusion proteins. FimH, CsgA, and PapG stimulated the release of 372–398 pg/mL IL-6; interestingly, FC and FCP stimulated the release of 464.79 pg/mL (p ≤ 0.018) and 521.24 pg/mL (p ≤ 0.002) IL-6, respectively. In addition, FC and FCP stimulated the release of 398.52 pg/mL (p ≤ 0.001) and 450.40 pg/mL (p ≤ 0.002) IL-8, respectively. High levels of IgA and IgG antibodies in human sera reacted against the fusion proteins, and under identical conditions, low levels of IgA and IgG antibodies were detected in human urine. Rabbit polyclonal antibodies

  6. Gelation Mechanisms and Characterization of Electrochemically Generated Protein Films at Metal Interfaces

    Science.gov (United States)

    Martin, Elizabeth J.

    Although the electrochemical behavior of metals used in orthopedic implants has been studied extensively, the material interactions with proteins during corrosion processes remains poorly understood. Some studies suggest that metal-protein interactions accelerate corrosion, while others suggest that proteins protect the material from degradation. Corrosion of implant materials is a major concern due to the metal ion release that can sometimes cause adverse local tissue reactions and ultimately, failure of the implant. The initial purpose of this research was therefore to study the corrosion behavior of CoCrMo, an alloy commonly used in hip replacements, with a quartz crystal microbalance (QCM) in physiologically relevant media. The QCM enables in situ characterization of surface changes accompanying corrosion and is sensitive to viscoelastic effects at its surface. Results of QCM studies in proteinaceous media showed film deposition on the alloy surface under electrochemical conditions that otherwise produced mass loss if proteins were not present in the electrolyte. Additional studies on pure Co, Cr, and Mo demonstrated that the protein films also form on Mo surfaces after a release of molybdate ions, suggesting that these ions are essential for film formation. The electrochemically generated protein films are reminiscent of carbonaceous films that form on implant surfaces in vivo, therefore a second goal of the research was to delineate mechanisms that cause the films to form. In the second stage of this research, electrochemical QCM tests were conducted on models of the CoCrMo system consisting of Cr electrodes in proteinaceous or polymeric media containing dissolved molybdate ions. Studies indicated that films can be generated through electrochemical processes so long as both amine functional groups and molybdate ions are present in the electrolyte solution. These results suggest that the films form due to an ionic cross-linking reaction between the positively

  7. Protein preconcentration using nanofractures generated by nanoparticle-assisted electric breakdown at junction gaps.

    Directory of Open Access Journals (Sweden)

    Chun-Ping Jen

    Full Text Available Sample preconcentration is an important step that increases the accuracy of subsequent detection, especially for samples with extremely low concentrations. Due to the overlapping of electrical double layers in the nanofluidic channel, the concentration polarization effect can be generated by applying an electric field. Therefore, a nonlinear electrokinetic flow is induced, which results in the fast accumulation of proteins in front of the induced ionic depletion zone, the so-called exclusion-enrichment effect. Nanofractures were created in this work to preconcentrate proteins via the exclusion-enrichment effect. The protein sample was driven by electroosmotic flow and accumulated at a specific location. The preconcentration chip for proteins was fabricated using simple standard soft lithography with a polydimethylsiloxane replica. Nanofractures were formed by utilizing nanoparticle-assisted electric breakdown. The proposed method for nanofracture formation that utilizes nanoparticle deposition at the junction gap between microchannels greatly decreases the required electric breakdown voltage. The experimental results indicate that a protein sample with an extremely low concentration of 1 nM was concentrated to 1.5×10(4-fold in 60 min using the proposed chip.

  8. Generation of diploid Pichia pastoris strains by mating and their application for recombinant protein production

    Directory of Open Access Journals (Sweden)

    Chen Ming-Tang

    2012-07-01

    Full Text Available Abstract Background Yeast mating provides an efficient means for strain and library construction. However, biotechnological applications of mating in the methylotrophic yeast Pichia pastoris have been hampered because of concerns about strain stability of P. pastoris diploids. The aim of the study reported here is to investigate heterologous protein expression in diploid P. pastoris strains and to evaluate diploid strain stability using high cell density fermentation processes. Results By using a monoclonal antibody as a target protein, we demonstrate that recombinant protein production in both wild-type and glycoengineered P. pastoris diploids is stable and efficient during a nutrient rich shake flask cultivation. When diploid strains were cultivated under bioreactor conditions, sporulation was observed. Nevertheless, both wild-type and glycoengineered P. pastoris diploids showed robust productivity and secreted recombinant antibody of high quality. Specifically, the yeast culture maintained a diploid state for 240 h post-induction phase while protein titer and N-linked glycosylation profiles were comparable to that of a haploid strain expressing the same antibody. As an application of mating, we also constructed an antibody display library and used mating to generate novel full-length antibody sequences. Conclusions To the best of our knowledge, this study reports for the first time a comprehensive characterization of recombinant protein expression and fermentation using diploid P. pastoris strains. Data presented here support the use of mating for various applications including strain consolidation, variable-region glycosylation antibody display library, and process optimization.

  9. CentrosomeDB: a new generation of the centrosomal proteins database for Human and Drosophila melanogaster.

    Science.gov (United States)

    Alves-Cruzeiro, Joao Miguel da Conceiçao; Nogales-Cadenas, Rubén; Pascual-Montano, Alberto Domingo

    2014-01-01

    We present the second generation of centrosomeDB, available online at http://centrosome.cnb.csic.es, with a significant expansion of 1357 human and drosophila centrosomal genes and their corresponding information. The centrosome of animal cells takes part in important biological processes such as the organization of the interphase microtubule cytoskeleton and the assembly of the mitotic spindle. The active research done during the past decades has produced lots of data related to centrosomal proteins. Unfortunately, the accumulated data are dispersed among diverse and heterogeneous sources of information. We believe that the availability of a repository collecting curated evidences of centrosomal proteins would constitute a key resource for the scientific community. This was our first motivation to introduce CentrosomeDB in NAR database issue in 2009, collecting a set of human centrosomal proteins that were reported in the literature and other sources. The intensive use of this resource during these years has encouraged us to present this new expanded version. Using our database, the researcher is offered the possibility to study the evolution, function and structure of the centrosome. We have compiled information from many sources, including Gene Ontology, disease-association, single nucleotide polymorphisms and associated gene expression experiments. Special interest has been paid to protein-protein interaction.

  10. Chiral vibrational structures of proteins at interfaces probed by sum frequency generation spectroscopy.

    Science.gov (United States)

    Fu, Li; Wang, Zhuguang; Yan, Elsa C Y

    2011-01-01

    We review the recent development of chiral sum frequency generation (SFG) spectroscopy and its applications to study chiral vibrational structures at interfaces. This review summarizes observations of chiral SFG signals from various molecular systems and describes the molecular origins of chiral SFG response. It focuses on the chiral vibrational structures of proteins and presents the chiral SFG spectra of proteins at interfaces in the C-H stretch, amide I, and N-H stretch regions. In particular, a combination of chiral amide I and N-H stretches of the peptide backbone provides highly characteristic vibrational signatures, unique to various secondary structures, which demonstrate the capacity of chiral SFG spectroscopy to distinguish protein secondary structures at interfaces. On the basis of these recent developments, we further discuss the advantages of chiral SFG spectroscopy and its potential application in various fields of science and technology. We conclude that chiral SFG spectroscopy can be a new approach to probe chiral vibrational structures of protein at interfaces, providing structural and dynamic information to study in situ and in real time protein structures and dynamics at interfaces.

  11. Next Generation Protein Interactomes for Plant Systems Biology and Biomass Feedstock Research

    Energy Technology Data Exchange (ETDEWEB)

    Ecker, Joseph Robert [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; Trigg, Shelly [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; Univ. of California, San Diego, CA (United States). Biological Sciences Dept.; Garza, Renee [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; Song, Haili [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; MacWilliams, Andrew [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; Nery, Joseph [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; Reina, Joaquin [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; Bartlett, Anna [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; Castanon, Rosa [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; Goubil, Adeline [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; Feeney, Joseph [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; O' Malley, Ronan [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; Huang, Shao-shan Carol [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; Zhang, Zhuzhu [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.; Galli, Mary [The Salk Inst. for Biological Studies, La Jolla, CA (United States). Genome Analysis and Plant Biology Lab.

    2016-11-30

    Biofuel crop cultivation is a necessary step in heading towards a sustainable future, making their genomic studies a priority. While technology platforms that currently exist for studying non-model crop species, like switch-grass or sorghum, have yielded large quantities of genomic and expression data, still a large gap exists between molecular mechanism and phenotype. The aspect of molecular activity at the level of protein-protein interactions has recently begun to bridge this gap, providing a more global perspective. Interactome analysis has defined more specific functional roles of proteins based on their interaction partners, neighborhoods, and other network features, making it possible to distinguish unique modules of immune response to different plant pathogens(Jiang, Dong, and Zhang 2016). As we work towards cultivating heartier biofuel crops, interactome data will lead to uncovering crop-specific defense and development networks. However, the collection of protein interaction data has been limited to expensive, time-consuming, hard-to-scale assays that mostly require cloned ORF collections. For these reasons, we have successfully developed a highly scalable, economical, and sensitive yeast two-hybrid assay, ProCREate, that can be universally applied to generate proteome-wide primary interactome data. ProCREate enables en masse pooling and massively paralleled sequencing for the identification of interacting proteins by exploiting Cre-lox recombination. ProCREate can be used to screen ORF/cDNA libraries from feedstock plant tissues. The interactome data generated will yield deeper insight into many molecular processes and pathways that can be used to guide improvement of feedstock productivity and sustainability.

  12. Protein Nano-Object Integrator (ProNOI for generating atomic style objects for molecular modeling

    Directory of Open Access Journals (Sweden)

    Smith Nicholas

    2012-12-01

    Full Text Available Abstract Background With the progress of nanotechnology, one frequently has to model biological macromolecules simultaneously with nano-objects. However, the atomic structures of the nano objects are typically not available or they are solid state entities. Because of that, the researchers have to investigate such nano systems by generating models of the nano objects in a manner that the existing software be able to carry the simulations. In addition, it should allow generating composite objects with complex shape by combining basic geometrical figures and embedding biological macromolecules within the system. Results Here we report the Protein Nano-Object Integrator (ProNOI which allows for generating atomic-style geometrical objects with user desired shape and dimensions. Unlimited number of objects can be created and combined with biological macromolecules in Protein Data Bank (PDB format file. Once the objects are generated, the users can use sliders to manipulate their shape, dimension and absolute position. In addition, the software offers the option to charge the objects with either specified surface or volumetric charge density and to model them with user-desired dielectric constants. According to the user preference, the biological macromolecule atoms can be assigned charges and radii according to four different force fields: Amber, Charmm, OPLS and PARSE. The biological macromolecules and the atomic-style objects are exported as a position, charge and radius (PQR file, or if a default dielectric constant distribution is not selected, it is exported as a position, charge, radius and epsilon (PQRE file. As illustration of the capabilities of the ProNOI, we created a composite object in a shape of a robot, aptly named the Clemson Robot, whose parts are charged with various volumetric charge densities and holds the barnase-barstar protein complex in its hand. Conclusions The Protein Nano-Object Integrator (ProNOI is a convenient tool for

  13. Protein Nano-Object Integrator (ProNOI) for generating atomic style objects for molecular modeling.

    Science.gov (United States)

    Smith, Nicholas; Campbell, Brandon; Li, Lin; Li, Chuan; Alexov, Emil

    2012-12-05

    With the progress of nanotechnology, one frequently has to model biological macromolecules simultaneously with nano-objects. However, the atomic structures of the nano objects are typically not available or they are solid state entities. Because of that, the researchers have to investigate such nano systems by generating models of the nano objects in a manner that the existing software be able to carry the simulations. In addition, it should allow generating composite objects with complex shape by combining basic geometrical figures and embedding biological macromolecules within the system. Here we report the Protein Nano-Object Integrator (ProNOI) which allows for generating atomic-style geometrical objects with user desired shape and dimensions. Unlimited number of objects can be created and combined with biological macromolecules in Protein Data Bank (PDB) format file. Once the objects are generated, the users can use sliders to manipulate their shape, dimension and absolute position. In addition, the software offers the option to charge the objects with either specified surface or volumetric charge density and to model them with user-desired dielectric constants. According to the user preference, the biological macromolecule atoms can be assigned charges and radii according to four different force fields: Amber, Charmm, OPLS and PARSE. The biological macromolecules and the atomic-style objects are exported as a position, charge and radius (PQR) file, or if a default dielectric constant distribution is not selected, it is exported as a position, charge, radius and epsilon (PQRE) file. As illustration of the capabilities of the ProNOI, we created a composite object in a shape of a robot, aptly named the Clemson Robot, whose parts are charged with various volumetric charge densities and holds the barnase-barstar protein complex in its hand. The Protein Nano-Object Integrator (ProNOI) is a convenient tool for generating atomic-style nano shapes in conjunction with

  14. The role of the carbohydrate recognition domain of placental protein 13 (PP13 in pregnancy evaluated with recombinant PP13 and the DelT221 PP13 variant.

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    Marei Sammar

    Full Text Available Placental protein 13 (PP13, a placenta specific protein, is reduced in the first trimester of pregnancy in women who subsequently develop preeclampsia. A naturally occurring PP13 deletion of thymidine at position 221 (DelT221 or truncated variant is associated with increased frequency of severe preeclampsia. In this study we compared the full length (wildtype PP13 and the truncated variant.Full length PP13 or its DelT221 variant were cloned, expressed and purified from E-Coli. Both variants were administrated into pregnant rats at day 8 of pregnancy for slow release (>5 days through osmotic pumps and rat blood pressure was measured. Animals were sacrificed at day 15 or day 21 and their utero-placental vasculature was examined.The DelT221 variant (11 kDA lacked exon 4 and a part of exon 3, and is short of 2 amino acids involved in the carbohydrate (CRD binding of the wildtype (18 kDA. Unlike the wildtype PP13, purification of DelT221 variant required special refolding. PP13 specific poly- clonal antibodies recognized both PP13 and DelT221 but PP13 specific monoclonal antibodies recognized only the wildtype, indicating the loss of major epitopes. Wildtype PP13 mRNA and its respective proteins were both lower in PE patients compared to normal pregnancies. The DelT221 mutant was not found in a large Caucasian cohort. Pregnant rats exposed to wildtype or DelT221 PP13 variants had significantly lower blood pressure compared to control. The wildtype but not the DelT221 mutant caused extensive vein expansion.This study revealed the importance of PP13 in regulating blood pressure and expanding the utero-placental vasculature in pregnant rats. PP13 mutant lacking amino acids of the PP13 CRD domain fails to cause vein expansion but did reduce blood pressure. The study provides a basis for replenishing patients at risk for preeclampsia by the full length but not the truncated PP13.

  15. Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation

    Science.gov (United States)

    Stopa, Jack D.; Neuberg, Donna; Puligandla, Maneka; Furie, Bruce; Zwicker, Jeffrey I.

    2017-01-01

    BACKGROUND: Protein disulfide isomerase (PDI) is required for thrombus formation. We previously demonstrated that glycosylated quercetin flavonoids such as isoquercetin inhibit PDI activity and thrombus formation in animal models, but whether extracellular PDI represents a viable anticoagulant target in humans and how its inhibition affects blood coagulation remain unknown. METHODS: We evaluated effects of oral administration of isoquercetin on platelet-dependent thrombin generation in healthy subjects and patients with persistently elevated anti-phospholipid antibodies. RESULTS: Following oral administration of 1,000 mg isoquercetin to healthy adults, the measured peak plasma quercetin concentration (9.2 μM) exceeded its IC50 for inhibition of PDI by isoquercetin in vitro (2.5 ± 0.4 μM). Platelet-dependent thrombin generation decreased by 51% in the healthy volunteers compared with baseline (P = 0.0004) and by 64% in the anti-phospholipid antibody cohort (P = 0.015) following isoquercetin ingestion. To understand how PDI affects thrombin generation, we evaluated substrates of PDI identified using an unbiased mechanistic-based substrate trapping approach. These studies identified platelet factor V as a PDI substrate. Isoquercetin blocked both platelet factor Va and thrombin generation with an IC50 of ~5 μM. Inhibition of PDI by isoquercetin ingestion resulted in a 53% decrease in the generation of platelet factor Va (P = 0.001). Isoquercetin-mediated inhibition was reversed with addition of exogenous factor Va. CONCLUSION: These studies show that oral administration of isoquercetin inhibits PDI activity in plasma and diminishes platelet-dependent thrombin generation predominantly by blocking the generation of platelet factor Va. These pharmacodynamic and mechanistic observations represent an important step in the development of a novel class of antithrombotic agents targeting PDI. TRIAL REGISTRATION: Clinicaltrials.gov (NCT01722669) FUNDING: National Heart

  16. Using protein misfolding cyclic amplification generates a highly neurotoxic PrP dimer causing neurodegeneration.

    Science.gov (United States)

    Yang, XiuJin; Yang, LiFeng; Zhou, XiangMei; Khan, Sher Hayat; Wang, HuiNuan; Yin, XiaoMin; Yuan, Zhen; Song, ZhiQi; Wu, WenYu; Zhao, DeMing

    2013-11-01

    Under the "protein-only" hypothesis, prion-based diseases are proposed to result from an infectious agent that is an abnormal isoform of the prion protein in the scrapie form, PrP(Sc). However, since PrP(Sc) is highly insoluble and easily aggregates in vivo, this view appears to be overly simplistic, implying that the presence of PrP(Sc) may indirectly cause neurodegeneration through its intermediate soluble form. We generated a neurotoxic PrP dimer with partial pathogenic characteristics of PrP(Sc) by protein misfolding cyclic amplification in the presence of 1-palmitoyl-2-oleoylphosphatidylglycerol consisting of recombinant hamster PrP (23-231). After intracerebral injection of the PrP dimer, wild-type hamsters developed signs of neurodegeneration. Clinical symptoms, necropsy findings, and histopathological changes were very similar to those of transmissible spongiform encephalopathies. Additional investigation showed that the toxicity is primarily related to cellular apoptosis. All results suggested that we generated a new neurotoxic form of PrP, PrP dimer, which can cause neurodegeneration. Thus, our study introduces a useful model for investigating PrP-linked neurodegenerative mechanisms.

  17. Immunoproteasome LMP2 60HH variant alters MBP epitope generation and reduces the risk to develop multiple sclerosis in Italian female population.

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    Michele Mishto

    Full Text Available BACKGROUND: Albeit several studies pointed out the pivotal role that CD4+T cells have in Multiple Sclerosis, the CD8+ T cells involvement in the pathology is still in its early phases of investigation. Proteasome degradation is the key step in the production of MHC class I-restricted epitopes and therefore its activity could be an important element in the activation and regulation of autoreactive CD8+ T cells in Multiple Sclerosis. METHODOLOGY/PRINCIPAL FINDINGS: Immunoproteasomes and PA28-alphabeta regulator are present in MS affected brain area and accumulated in plaques. They are expressed in cell types supposed to be involved in MS development such as neurons, endothelial cells, oligodendrocytes, macrophages/macroglia and lymphocytes. Furthermore, in a genetic study on 1262 Italian MS cases and 845 controls we observed that HLA-A*02+ female subjects carrying the immunoproteasome LMP2 codon 60HH variant have a reduced risk to develop MS. Accordingly, immunoproteasomes carrying the LMP2 60H allele produce in vitro a lower amount of the HLA-A*0201 restricted immunodominant epitope MBP(111-119. CONCLUSION/SIGNIFICANCE: The immunoproteasome LMP2 60HH variant reduces the risk to develop MS amongst Italian HLA-A*02+ females. We propose that such an effect is mediated by the altered proteasome-dependent production of a specific MBP epitope presented on the MHC class I. Our observations thereby support the hypothesis of an involvement of immunoproteasome in the MS pathogenesis.

  18. A Remodeled Protein Arginine Methyltransferase 1 (PRMT1) Generates Symmetric Dimethylarginine*

    Science.gov (United States)

    Gui, Shanying; Gathiaka, Symon; Li, Jun; Qu, Jun; Acevedo, Orlando; Hevel, Joan M.

    2014-01-01

    Protein arginine methylation is emerging as a significant post-translational modification involved in various cell processes and human diseases. As the major arginine methylation enzyme, protein arginine methyltransferase 1 (PRMT1) strictly generates monomethylarginine and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA). The two types of dimethylarginines can lead to distinct biological outputs, as highlighted in the PRMT-dependent epigenetic control of transcription. However, it remains unclear how PRMT1 product specificity is regulated. We discovered that a single amino acid mutation (Met-48 to Phe) in the PRMT1 active site enables PRMT1 to generate both ADMA and SDMA. Due to the limited amount of SDMA formed, we carried out quantum mechanical calculations to determine the free energies of activation of ADMA and SDMA synthesis. Our results indicate that the higher energy barrier of SDMA formation (ΔΔG‡ = 3.2 kcal/mol as compared with ADMA) may explain the small amount of SDMA generated by M48F-PRMT1. Our study reveals unique energetic challenges for SDMA-forming methyltransferases and highlights the exquisite control of product formation by active site residues in the PRMTs. PMID:24478314

  19. Generation of glycosylphosphatidylinositol anchor protein-deficient blood cells from human induced pluripotent stem cells.

    Science.gov (United States)

    Yuan, Xuan; Braunstein, Evan M; Ye, Zhaohui; Liu, Cyndi F; Chen, Guibin; Zou, Jizhong; Cheng, Linzhao; Brodsky, Robert A

    2013-11-01

    PIG-A is an X-linked gene required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; thus, PIG-A mutant cells have a deficiency or absence of all GPI-anchored proteins (GPI-APs). Acquired mutations in hematopoietic stem cells result in the disease paroxysmal nocturnal hemoglobinuria, and hypomorphic germline PIG-A mutations lead to severe developmental abnormalities, seizures, and early death. Human induced pluripotent stem cells (iPSCs) can differentiate into cell types derived from all three germ layers, providing a novel developmental system for modeling human diseases. Using PIG-A gene targeting and an inducible PIG-A expression system, we have established, for the first time, a conditional PIG-A knockout model in human iPSCs that allows for the production of GPI-AP-deficient blood cells. PIG-A-null iPSCs were unable to generate hematopoietic cells or any cells expressing the CD34 marker and were defective in generating mesodermal cells expressing KDR/VEGFR2 (kinase insert domain receptor) and CD56 markers. In addition, PIG-A-null iPSCs had a block in embryonic development prior to mesoderm differentiation that appears to be due to defective signaling through bone morphogenetic protein 4. However, early inducible PIG-A transgene expression allowed for the generation of GPI-AP-deficient blood cells. This conditional PIG-A knockout model should be a valuable tool for studying the importance of GPI-APs in hematopoiesis and human development.

  20. A genetic variant of the CAPN10 gene in Mexican subjects with dyslipidemia is associated with increased HDL-cholesterol concentrations after the consumption of a soy protein and soluble fiber dietary portfolio

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    Martha Guevara-Cruz

    Full Text Available Dyslipidemia is a major public health problem, and therefore, it is important to develop dietary strategies to diminish the prevalence of this disorder. It was recently reported that diet may play an important role in triggering insulin resistance by interacting with genetic variants at the CAPN10 gene locus in patients with metabolic syndrome. Nonetheless, it remains unknown whether genetic variants of genes involved in the development of type 2 diabetes are associated with variations in high-density lipoprotein cholesterol (HDL-C. The study used a single-center, prospective, cohort design. Here, we assessed the effect of four variants of the CAPN10 gene on HDL-C levels in response to a soy protein and soluble fiber dietary portfolio in subjects with dyslipidemia. In 31 Mexican dyslipidemic individuals, we analyzed four CAPN10 gene variants (rs5030952, rs2975762, rs3792267, and rs2975760 associated with type 2 diabetes. Subjects with the GG genotype of the rs2975762 variant of the CAPN10 gene were better responders to dietary intervention, showing increased HDL-C concentrations from the first month of treatment. HDL-C concentrations in participants with the wild type genotype increased by 17.0%, whereas the HDL-C concentration in subjects with the variant genotypes increased by only 3.22% (p = 0.03; the low-density lipoprotein cholesterol levels of GG carriers tended to decrease (-12.6%. These results indicate that Mexican dyslipidemic carriers of the rs2975762-GG genotype are better responders to this dietary intervention.

  1. Generating site-specifically modified proteins via a versatile and stable nucleophilic carbon ligation.

    Science.gov (United States)

    Kudirka, Romas; Barfield, Robyn M; McFarland, Jesse; Albers, Aaron E; de Hart, Gregory W; Drake, Penelope M; Holder, Patrick G; Banas, Stefanie; Jones, Lesley C; Garofalo, Albert W; Rabuka, David

    2015-02-19

    There is a need for facile chemistries that allow for chemo- and regioselectivity in bioconjugation reactions. To address this need, we are pioneering site-specific bioconjugation methods that use formylglycine as a bioorthogonal handle on a protein surface. Here we introduce aldehyde-specific bioconjugation chemistry, the trapped-Knoevenagel ligation. The speed and stability of the trapped-Knoevenagel ligation further advances the repertoire of aldehyde-based bioconjugations and expands the toolbox for site-specific protein modifications. The trapped-Knoevenagel ligation reaction can be run at near neutral pH in the absence of catalysts to produce conjugates that are stable under physiological conditions. Using this new ligation, we generated an antibody-drug conjugate that demonstrates excellent efficacy in vitro and in vivo.

  2. Generation of a Functionally Distinct Rhizopus oryzae Lipase through Protein Folding Memory.

    Science.gov (United States)

    Satomura, Atsushi; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2015-01-01

    Rhizopus oryzae lipase (ROL) has a propeptide at its N-terminus that functions as an intramolecular chaperone and facilitates the folding of mature ROL (mROL). In this study, we successfully generated a functionally distinct imprinted mROL (mROLimp) through protein folding memory using a mutated propeptide. The mutated propeptide left its structural memory on mROL and produced mROLimp that exhibited different substrate specificities compared with mROLWT (prepared from the wild type propeptide), although the amino acid sequences of both mROLs were the same. mROLimp showed a preference for substrates with medium chain-length acyl groups and, noticeably, recognized a peptidase-specific substrate. In addition, ROLimp was more stable than mROLWT. These results strongly suggest that proteins with identical amino acid sequences can fold into different conformations and that mutations in intramolecular chaperones can dynamically induce changes in enzymatic activity.

  3. Insulin-like growth factor 2 (IGF2 ) and IGF-binding protein 1 (IGFBP1) gene variants are associated with overfeeding-induced metabolic changes.

    Science.gov (United States)

    Ukkola, O; Sun, G; Bouchard, C

    2001-12-01

    The aim of this study was to investigate the role of insulin-like growth factor 1 (IGF1), IGF2, IGF binding protein 1 (IGFBP1) and IGFBP3 gene variants on the metabolic changes observed in response to a 100-day overfeeding protocol conducted with 12 pairs of monozygotic twins. Genotyping was done by PCR-RFLP and DNA sequencer methods. Body fat measurements included hydrodensitometry and abdominal fat from computed tomography. Plasma glucose and insulin during fasting and in response to an OGTT were assayed. Plasma lipids were measured enzymatically. In response to caloric surplus, fasting plasma insulin (p < 0.05) and OGTT insulin (p = 0.004) but not glucose area, increased more among the subjects with IGF2 Apa I GG (n = 12) than those with AA + AG (n = 12). The changes were independent of changes in total fatness. The subjects with IGFBP1 Bgl II AA (n = 8) showed greater increases in abdominal visceral fat (p < 0.01), OGTT insulin area (p = 0.05) and total cholesterol (p < 0.03) with overfeeding than the subjects with AG + GG (n = 16). IGFBP3 Nde I and the IGF1 (CT)n markers were not associated with responsiveness to overfeeding. Insulin sensitivity decreased in the subjects with IGF2 Apa I GG and the subjects with IGFBP1 Bgl II AA showed an accumulation of abdominal visceral fat and the early symptoms of the metabolic syndrome after long-term caloric surplus. Genetic variation at the IGF2 and IGFBP1 loci could be among the factors responsible for the inter-individual differences observed in the response to long-term alterations in energy balance and should be further investigated in larger cohorts.

  4. Assessing the role of insulin‐like growth factors and binding proteins in prostate cancer using Mendelian randomization: Genetic variants as instruments for circulating levels

    Science.gov (United States)

    Bonilla, Carolina; Lewis, Sarah J.; Rowlands, Mari‐Anne; Gaunt, Tom R.; Davey Smith, George; Gunnell, David; Palmer, Tom; Donovan, Jenny L.; Hamdy, Freddie C.; Neal, David E.; Eeles, Rosalind; Easton, Doug; Kote‐Jarai, Zsofia; Al Olama, Ali Amin; Benlloch, Sara; Muir, Kenneth; Giles, Graham G.; Wiklund, Fredrik; Grönberg, Henrik; Haiman, Christopher A.; Schleutker, Johanna; Nordestgaard, Børge G.; Travis, Ruth C.; Pashayan, Nora; Khaw, Kay‐Tee; Stanford, Janet L.; Blot, William J.; Thibodeau, Stephen; Maier, Christiane; Kibel, Adam S; Cybulski, Cezary; Cannon‐Albright, Lisa; Brenner, Hermann; Park, Jong; Kaneva, Radka; Batra, Jyotsna; Teixeira, Manuel R.; Pandha, Hardev; Lathrop, Mark; Holly, Jeff M. P.

    2016-01-01

    Circulating insulin‐like growth factors (IGFs) and their binding proteins (IGFBPs) are associated with prostate cancer. Using genetic variants as instruments for IGF peptides, we investigated whether these associations are likely to be causal. We identified from the literature 56 single nucleotide polymorphisms (SNPs) in the IGF axis previously associated with biomarker levels (8 from a genome‐wide association study [GWAS] and 48 in reported candidate genes). In ∼700 men without prostate cancer and two replication cohorts (N ∼ 900 and ∼9,000), we examined the properties of these SNPS as instrumental variables (IVs) for IGF‐I, IGF‐II, IGFBP‐2 and IGFBP‐3. Those confirmed as strong IVs were tested for association with prostate cancer risk, low (< 7) vs. high (≥ 7) Gleason grade, localised vs. advanced stage, and mortality, in 22,936 controls and 22,992 cases. IV analysis was used in an attempt to estimate the causal effect of circulating IGF peptides on prostate cancer. Published SNPs in the IGFBP1/IGFBP3 gene region, particularly rs11977526, were strong instruments for IGF‐II and IGFBP‐3, less so for IGF‐I. Rs11977526 was associated with high (vs. low) Gleason grade (OR per IGF‐II/IGFBP‐3 level‐raising allele 1.05; 95% CI: 1.00, 1.10). Using rs11977526 as an IV we estimated the causal effect of a one SD increase in IGF‐II (∼265 ng/mL) on risk of high vs. low grade disease as 1.14 (95% CI: 1.00, 1.31). Because of the potential for pleiotropy of the genetic instruments, these findings can only causally implicate the IGF pathway in general, not any one specific biomarker. PMID:27225428

  5. Human T-cell recognition of synthetic peptides representing conserved and variant sequences from the merozoite surface protein 2 of Plasmodium falciparum.

    Science.gov (United States)

    Theander, T G; Hviid, L; Dodoo, D; Afari, E A; Jensen, J B; Rzepczyk, C M

    1997-06-01

    Merozoite surface protein 2 (MSP2) is a malaria vaccine candidate currently undergoing clinical trials. We analyzed the peripheral blood mononuclear cell (PBMC) response to synthetic peptides corresponding to conserved and variant regions of the FCQ-27 allelic form of MSP2 in Ghanaian individuals from an area of hyperendemic malaria transmission and in Danes without exposure to malaria. PBMC from 20-39% of Ghanaians responded to each of the peptides by proliferation and 29-36% had PBMC which produced interferon-gamma (IFN-gamma) in response to peptide stimulation. In Danes, there was no proliferation to two of the peptides and only PBMC from 5% of the individuals proliferated to the other three peptides. IFN-gamma production was not detected to any peptide. In both Danes and Ghanaians in only a few instances was IL-4 detected in the PBMC cultures. Overall PBMC from 79% of the Ghanaians responded by proliferation and/or cytokine secretion to at least one of three peptides tested, whereas responses were only observed in 14% of Danes (P = 0.002). These data suggest that the Ghanaians had expanded peripheral blood T-cell populations recognizing the peptides as a result of natural infection. The findings are encouraging for the development of a vaccine based on these T-epitope containing regions of MSP2, as the peptides were broadly recognized suggesting that they can bind to diverse HLA alleles and also because they include conserved MSP2 sequences. Immunisation with a vaccine construct incorporating the sequences present in these peptides could thus be expected to be immunogenic in a high percentage of individuals and lead to the establishment of memory T-cells, which can be boosted through natural infection.

  6. Generation and characterization of a highly effective protein substrate for analysis of FLT3 activity

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    Chen Yun

    2012-07-01

    Full Text Available Abstract Background Gain-of-function mutations of tyrosine kinase FLT3 are frequently found in acute myeloid leukemia (AML. This has made FLT3 an important marker for disease diagnosis and a highly attractive target for therapeutic drug development. This study is intended to generate a sensitive substrate for assays of the FLT3 enzymatic activity. Methods We expressed in Escherichia coli cells a glutathione S-transferase (GST fusion protein designated GST-FLT3S, which contains a peptide sequence derived from an autophosphorylation site of FLT3. The protein was used to analyze tyrosine kinase activity of baculovirus-expressed FLT3 and crude cell extracts of bone marrow cells from AML patients. It was also employed to perform FLT3 kinase assays for FLT3 inhibitor screening. Results GST-FLT3S in solution or on beads was strongly phosphorylated by recombinant proteins carrying the catalytic domain of wild type FLT3 and FLT3D835 mutants, with the latter exhibiting much higher activity and efficiency. GST-FLT3S was also able to detect elevated tyrosine kinase activity in bone marrow cell extracts from AML patients. A small-scale inhibitor screening led to identification of several potent inhibitors of wild type and mutant forms of FLT3. Conclusions GST-FLT3S is a sensitive protein substrate for FLT3 assays. It may find applications in diagnosis of diseases related to abnormal FLT3 activity and in inhibitor screening for drug development.

  7. Intra-generational protein malnutrition impairs temporal astrogenesis in rat brain.

    Science.gov (United States)

    Naik, Aijaz Ahmad; Patro, Nisha; Seth, Pankaj; Patro, Ishan K

    2017-07-15

    The lack of information on astrogenesis following stressor effect, notwithstanding the imperative roles of astroglia in normal physiology and pathophysiology, incited us to assess temporal astrogenesis and astrocyte density in an intra-generational protein malnutrition (PMN) rat model. Standard immunohistochemical procedures for glial lineage markers and their intensity measurements, and qRT-PCR studies, were performed to reveal the spatio-temporal origin and density of astrocytes. Reduced A2B5+ glia restricted precursor population in ventricles and caused poor dissemination to cortex at embryonic days (E)11-14, and low BLBP+ secondary radial glia in the subventricular zone (SVZ) of E16 low protein (LP) brains reflect compromised progenitor pooling. Contrary to large-sized BLBP+ gliospheres in high protein (HP) brains at E16, small gliospheres and discrete BLBP+ cells in LP brains evidence loss of colonization and low proliferative potential. Delayed emergence of GFAP expression, precocious astrocyte maturation and significantly reduced astrocyte number suggest impaired temporal and compromised astrogenesis within LP-F1 brains. Our findings of protein deprivation induced impairments in temporal astrogenesis, compromised density and astrocytic dysfunction, strengthen the hypothesis of astrocytes as possible drivers of neurodevelopmental disorders. This study may increase our understanding of stressor-associated brain development, opening up windows for effective therapeutic interventions against debilitating neurodevelopmental disorders. © 2017. Published by The Company of Biologists Ltd.

  8. Intra-generational protein malnutrition impairs temporal astrogenesis in rat brain

    Directory of Open Access Journals (Sweden)

    Aijaz Ahmad Naik

    2017-07-01

    Full Text Available The lack of information on astrogenesis following stressor effect, notwithstanding the imperative roles of astroglia in normal physiology and pathophysiology, incited us to assess temporal astrogenesis and astrocyte density in an intra-generational protein malnutrition (PMN rat model. Standard immunohistochemical procedures for glial lineage markers and their intensity measurements, and qRT-PCR studies, were performed to reveal the spatio-temporal origin and density of astrocytes. Reduced A2B5+ glia restricted precursor population in ventricles and caused poor dissemination to cortex at embryonic days (E11-14, and low BLBP+ secondary radial glia in the subventricular zone (SVZ of E16 low protein (LP brains reflect compromised progenitor pooling. Contrary to large-sized BLBP+ gliospheres in high protein (HP brains at E16, small gliospheres and discrete BLBP+ cells in LP brains evidence loss of colonization and low proliferative potential. Delayed emergence of GFAP expression, precocious astrocyte maturation and significantly reduced astrocyte number suggest impaired temporal and compromised astrogenesis within LP-F1 brains. Our findings of protein deprivation induced impairments in temporal astrogenesis, compromised density and astrocytic dysfunction, strengthen the hypothesis of astrocytes as possible drivers of neurodevelopmental disorders. This study may increase our understanding of stressor-associated brain development, opening up windows for effective therapeutic interventions against debilitating neurodevelopmental disorders.

  9. A mathematical model for generating bipartite graphs and its application to protein networks

    Energy Technology Data Exchange (ETDEWEB)

    Nacher, J C [Department of Complex Systems, Future University-Hakodate (Japan); Ochiai, T [Faculty of Engineering, Toyama Prefectural University (Japan); Hayashida, M; Akutsu, T [Bioinformatics Center, Institute for Chemical Research, Kyoto University (Japan)

    2009-12-04

    Complex systems arise in many different contexts from large communication systems and transportation infrastructures to molecular biology. Most of these systems can be organized into networks composed of nodes and interacting edges. Here, we present a theoretical model that constructs bipartite networks with the particular feature that the degree distribution can be tuned depending on the probability rate of fundamental processes. We then use this model to investigate protein-domain networks. A protein can be composed of up to hundreds of domains. Each domain represents a conserved sequence segment with specific functional tasks. We analyze the distribution of domains in Homo sapiens and Arabidopsis thaliana organisms and the statistical analysis shows that while (a) the number of domain types shared by k proteins exhibits a power-law distribution, (b) the number of proteins composed of k types of domains decays as an exponential distribution. The proposed mathematical model generates bipartite graphs and predicts the emergence of this mixing of (a) power-law and (b) exponential distributions. Our theoretical and computational results show that this model requires (1) growth process and (2) copy mechanism.

  10. Genetic Variants in the Bone Morphogenic Protein Gene Family Modify the Association between Residential Exposure to Traffic and Peripheral Arterial Disease

    Science.gov (United States)

    There is a growing literature indicating that genetic variants modify many of the associations between environmental exposures and clinical outcomes, potentially by increasing susceptibility to these exposures. However, genome-scale investigations of these interactions have been ...

  11. Generation of an affinity column for antibody purification by intein-mediated protein ligation.

    Science.gov (United States)

    Sun, Luo; Ghosh, Inca; Xu, Ming-Qun

    2003-11-01

    Coupling an antigenic peptide to a solid support is a crucial step in the affinity purification of a peptide-specific antibody. Conventional methods for generating reactive agarose, cellulose or other matrices for peptide conjugation are laborious and can result in a significant amount of chemical waste. In this report, we present a novel method for the facile production of a peptide affinity column by employing intein-mediated protein ligation (IPL) in conjunction with chitin affinity chromatography. A reactive thioester was generated at the C-terminal of the chitin binding domain (CBD) from the chitinase A1 of Bacillus circulans WL-2 by thiol-induced cleavage of the peptide bond between the CBD and a modified intein. Peptide epitopes possessing an N-terminal cysteine were ligated to the chitin bound CBD tag. We demonstrate that the resulting peptide columns permit the highly specific and efficient affinity purification of antibodies from animal sera.

  12. Protein disulfide bond generation in Escherichia coli DsbB–DsbA

    OpenAIRE

    Inaba, Kenji

    2008-01-01

    Protein disulfide bond formation is catalyzed by a series of Dsb enzymes present in the periplasm of Escherichia coli. The crystal structure of the DsbB–DsbA–ubiquinone ternary complex provided important insights into mechanisms of the de novo disulfide bond generation cooperated by DsbB and ubiquinone and of the disulfide bond shuttle from DsbB to DsbA. The structural basis for prevention of the crosstalk between the DsbA–DsbB oxidative and the DsbC–DsbD reductive pathways has also been prop...

  13. Generations.

    Science.gov (United States)

    Chambers, David W

    2005-01-01

    Groups naturally promote their strengths and prefer values and rules that give them an identity and an advantage. This shows up as generational tensions across cohorts who share common experiences, including common elders. Dramatic cultural events in America since 1925 can help create an understanding of the differing value structures of the Silents, the Boomers, Gen Xers, and the Millennials. Differences in how these generations see motivation and values, fundamental reality, relations with others, and work are presented, as are some applications of these differences to the dental profession.

  14. Red-backed vole brain promotes highly efficient in vitro amplification of abnormal prion protein from macaque and human brains infected with variant Creutzfeldt-Jakob disease agent.

    Science.gov (United States)

    Nemecek, Julie; Nag, Nabanita; Carlson, Christina M.; Schneider, Jay R.; Heisey, Dennis M.; Johnson, Christopher J.; Asher, David M.; Gregori, Luisa

    2013-01-01

    Rapid antemortem tests to detect individuals with transmissible spongiform encephalopathies (TSE) would contribute to public health. We investigated a technique known as protein misfolding cyclic amplification (PMCA) to amplify abnormal prion protein (PrPTSE) from highly diluted variant Creutzfeldt-Jakob disease (vCJD)-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrPTSE in tissues and blood. Macaque vCJD PrPTSE did not amplify using normal macaque brain homogenate as substrate (intraspecies PMCA). Next, we tested interspecies PMCA with normal brain homogenate of the southern red-backed vole (RBV), a close relative of the bank vole, seeded with macaque vCJD PrPTSE. The RBV has a natural polymorphism at residue 170 of the PrP-encoding gene (N/N, S/S, and S/N). We investigated the effect of this polymorphism on amplification of human and macaque vCJD PrPTSE. Meadow vole brain (170N/N PrP genotype) was also included in the panel of substrates tested. Both humans and macaques have the same 170S/S PrP genotype. Macaque PrPTSE was best amplified with RBV 170S/S brain, although 170N/N and 170S/N were also competent substrates, while meadow vole brain was a poor substrate. In contrast, human PrPTSE demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrPTSE was more permissive than human PrPTSE in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrPTSE from brains of humans and macaques with vCJD. PrPTSE signals were reproducibly detected by Western blot in dilutions through 10-12 of vCJD-infected 10% brain homogenates. This is the first report showing PrPTSE from vCJD-infected human and macaque brains efficiently amplified with RBV brain as the substrate. Based on our estimates, PMCA showed a sensitivity that might be sufficient to detect PrPTSE in v

  15. Red-backed vole brain promotes highly efficient in vitro amplification of abnormal prion protein from macaque and human brains infected with variant Creutzfeldt-Jakob disease agent.

    Directory of Open Access Journals (Sweden)

    Julie Nemecek

    Full Text Available Rapid antemortem tests to detect individuals with transmissible spongiform encephalopathies (TSE would contribute to public health. We investigated a technique known as protein misfolding cyclic amplification (PMCA to amplify abnormal prion protein (PrP(TSE from highly diluted variant Creutzfeldt-Jakob disease (vCJD-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrP(TSE in tissues and blood. Macaque vCJD PrP(TSE did not amplify using normal macaque brain homogenate as substrate (intraspecies PMCA. Next, we tested interspecies PMCA with normal brain homogenate of the southern red-backed vole (RBV, a close relative of the bank vole, seeded with macaque vCJD PrP(TSE. The RBV has a natural polymorphism at residue 170 of the PrP-encoding gene (N/N, S/S, and S/N. We investigated the effect of this polymorphism on amplification of human and macaque vCJD PrP(TSE. Meadow vole brain (170N/N PrP genotype was also included in the panel of substrates tested. Both humans and macaques have the same 170S/S PrP genotype. Macaque PrP(TSE was best amplified with RBV 170S/S brain, although 170N/N and 170S/N were also competent substrates, while meadow vole brain was a poor substrate. In contrast, human PrP(TSE demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrP(TSE was more permissive than human PrP(TSE in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrP(TSE from brains of humans and macaques with vCJD. PrP(TSE signals were reproducibly detected by Western blot in dilutions through 10⁻¹² of vCJD-infected 10% brain homogenates. This is the first report showing PrP(TSE from vCJD-infected human and macaque brains efficiently amplified with RBV brain as the substrate. Based on our estimates, PMCA showed a sensitivity that might be sufficient to detect Pr

  16. Immunogenic Domains and Secondary Structure of Escherichia coli Recombinant Secreted Protein Escherichia coli-Secreted Protein B

    Directory of Open Access Journals (Sweden)

    Roxane Maria Fontes Piazza

    2017-04-01

    Full Text Available Several pathogenic bacteria are able to induce the attaching and effacing (A/E lesion. The A/E lesion is caused by effector proteins, such as Escherichia coli-secreted protein B (EspB, responsible together with Escherichia coli-secreted protein D for forming a pore structure on the host cell, which allows the translocation of effector proteins. Different variants of this protein can be found in E. coli strains, and during natural infection or when this protein is injected, this leads to variant-specific production of antibodies, which may not be able to recognize other variants of this bacterial protein. Herein, we describe the production of a hybrid recombinant EspB toxin that comprises all known variants of this protein. This recombinant protein could be useful as an antigen for the production of antibodies with broad-range detection of EspB-bearing bacteria, or as an antigen that could be used in vaccine formulation to generate antibodies against different EspB variants, thereby increasing immunization potential. In addition, the recombinant protein allowed us to analyze its secondary structure, to propose the immunogenic regions of EspB variants, and also to characterize anti-EspB antibodies. Our results suggest that this hybrid protein or a protein composed of the conserved immunogenic regions could be used for a variety of clinical applications.

  17. Immunogenic Domains and Secondary Structure of Escherichia coli Recombinant Secreted Protein Escherichia coli-Secreted Protein B.

    Science.gov (United States)

    Caetano, Bruna Alves; Rocha, Letícia Barboza; Carvalho, Eneas; Piazza, Roxane Maria Fontes; Luz, Daniela

    2017-01-01

    Several pathogenic bacteria are able to induce the attaching and effacing (A/E) lesion. The A/E lesion is caused by effector proteins, such as Escherichia coli-secreted protein B (EspB), responsible together with Escherichia coli-secreted protein D for forming a pore structure on the host cell, which allows the translocation of effector proteins. Different variants of this protein can be found in E. coli strains, and during natural infection or when this protein is injected, this leads to variant-specific production of antibodies, which may not be able to recognize other variants of this bacterial protein. Herein, we describe the production of a hybrid recombinant EspB toxin that comprises all known variants of this protein. This recombinant protein could be useful as an antigen for the production of antibodies with broad-range detection of EspB-bearing bacteria, or as an antigen that could be used in vaccine formulation to generate antibodies against different EspB variants, thereby increasing immunization potential. In addition, the recombinant protein allowed us to analyze its secondary structure, to propose the immunogenic regions of EspB variants, and also to characterize anti-EspB antibodies. Our results suggest that this hybrid protein or a protein composed of the conserved immunogenic regions could be used for a variety of clinical applications.

  18. De novo generation of infectious prions with bacterially expressed recombinant prion protein.

    Science.gov (United States)

    Zhang, Zhihong; Zhang, Yi; Wang, Fei; Wang, Xinhe; Xu, Yuanyuan; Yang, Huaiyi; Yu, Guohua; Yuan, Chonggang; Ma, Jiyan

    2013-12-01

    The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.

  19. Protein kinase clk/STY is differentially regulated during erythroleukemia cell differentiation: a bias toward the skipped splice variant characterizes postcommitment stages

    Institute of Scientific and Technical Information of China (English)

    Ana GARC(I)A-SACRIST(A)N; María J.FERN(A)NDEZ-NESTOSA; Pablo HERN(A)NDEZ; Jorge B.SCHVARTZMAN; Dora B.KRIMER

    2005-01-01

    Clk/STY is a LAMMER protein kinase capable to phosphorylate serine/arginine-rich (SR) proteins that modulate premRNA splicing.Clk/STY alternative splicing generates transcripts encoding a full-length kinase and a truncated catalytically inactive protein.Here we showed that clk/STY,as well as other members of the family (e.g.clk2,clk3 and clk4),are up-regulated during HMBA-induced erythroleukemia cell differentiation.mRNAs coding for the full-length and the truncated forms were responsible for the overall increased expression.In clk/STY,however,a switch was observed for the ratio of the two alternative spliced products.In undifferentiated cells the full-length transcript was more abundant whereas the transcript encoding for the truncated form predominated at latter stages of differentiation.Surprisingly,overexpression of clk/STY did not alter the splicing switch upon differentiation in MEL cells.These results suggest that clk/STY might contribute to control erythroid differentiation by a mechanism that implicates a balance between these two isoforms.

  20. Establishment of Sf9 transformants constitutively expressing PBAN receptor (PBANR variants: application to functional evaluation

    Directory of Open Access Journals (Sweden)

    Jae Min Lee

    2012-04-01

    Full Text Available To facilitate further evaluation of pheromone biosynthesis activating neuropeptide receptor (PBANR functionality and regulation, we generated cultured insect cell lines constitutively expressing green fluorescent protein chimeras of the recently identified Bombyx mori PBANR (BommoPBANR and Pseudaletia separata PBANR (PsesePBANR variants. Fluorescent chimeras included the BommoPBANR-A, B, and C variants and the PsesePBANR-B and C variants. Cell lines expressing non-chimeric BommoPBANR-B and C variants were also generated. Functional evaluation of these transformed cell lines using confocal laser microscopy revealed that a Rhodamine Red-labeled PBAN derivative (RR-C10PBANR2K specifically co-localized with all of the respective PBANR variants at the plasma membrane. Near complete internalization of the fluorescent RR-C10PBANR2K ligand 30 min after binding was observed in all cell lines except those expressing the BommoPBANR-A variant, in which the ligand/receptor complex remained at the plasma membrane. Fluorescent Ca2+ imaging further showed that, unlike the BommoPBANR-B or BommoPBANR-C cell lines, RR-C10PBANR2K binding failed to mobilize extracellular Ca2+ in the BommoPBANR-A cell line even at concentrations of 10 M. These observations demonstrate a clear functional difference between the BommoPBANR-A variant and the BommoPBANR-B and –C variants in terms of receptor regulation and activation of downstream effector molecules. We also found that, contrary to previous reports, ligand-induced internalization of BommoPBANR-B and BommoPBANR-C in cell lines stably expressing these variants occurred in the absence of extracellular Ca2+.

  1. De Novo Generation of a Unique Cervid Prion Strain Using Protein Misfolding Cyclic Amplification

    Science.gov (United States)

    Meyerett-Reid, Crystal; Wyckoff, A. Christy; Spraker, Terry; Pulford, Bruce; Bender, Heather

    2017-01-01

    ABSTRACT Substantial evidence supports the hypothesis that prions are misfolded, infectious, insoluble, and protease-resistant proteins (PrPRES) devoid of instructional nucleic acid that cause transmissible spongiform encephalopathies (TSEs). Protein misfolding cyclic amplification (PMCA) has provided additional evidence that PrPRes acts as a template that can convert the normal cellular prion protein (PrPC) present in uninfected normal brain homogenate (NBH) into the infectious misfolded PrPRES isoform. Human PrPC has been shown to spontaneously convert to a misfolded pathological state causing sporadic Creutzfeldt-Jakob disease (sCJD). Several investigators have reported spontaneous generation of prions by in vitro assays, including PMCA. Here we tested the rate of de novo generation of cervid prions in our laboratory using our standard PMCA protocol and NBH from transgenic mice expressing cervid PrPC (TgCerPrP mice). We generated de novo prions in rounds 4, 5, and 7 at low cumulative rates of 1.6, 5.0, and 6.7%, respectively. The prions caused infectious chronic wasting disease (CWD) upon inoculation into normal uninfected TgCerPrP mice and displayed unique biochemical characteristics compared to other cervid prion strains. We conclude that PMCA of cervid PrPC from normal brain homogenate spontaneously generated a new cervid prion strain. These data support the potential for cervids to develop sporadic CWD. IMPORTANCE CWD is the only known TSE that affects free-ranging wildlife, specifically cervids such as elk, deer, moose, caribou, and reindeer. CWD has become endemic in both free-ranging and captive herds in North America, South Korea, and, most recently, northern Europe. The prion research community continues to debate the origins of CWD. Original foci of CWD emergence in Colorado and Wyoming coincident with the sheep TSE scrapie suggest that scrapie prions may have adapted to cervids to cause CWD. However, emerging evidence supports the idea that cervid Pr

  2. Non-coding keratin variants associate with liver fibrosis progression in patients with hemochromatosis.

    Directory of Open Access Journals (Sweden)

    Pavel Strnad

    Full Text Available BACKGROUND: Keratins 8 and 18 (K8/K18 are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis. METHODS: The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed. RESULTS: We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2% and non-coding heterozygous variants in 6 additional patients (3.7%. Two novel K8 variants (Q169E/R275W were found. K8 R341H was the most common amino-acid altering variant (4 patients, and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury. CONCLUSION: In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.

  3. Identification of immunogenic proteins and generation of antibodies against Salmonella Typhimurium using phage display

    Directory of Open Access Journals (Sweden)

    Meyer Torsten

    2012-06-01

    Full Text Available Abstract Background Solely in Europoe, Salmonella Typhimurium causes more than 100,000 infections per year. Improved detection of livestock colonised with S. Typhimurium is necessary to prevent foodborne diseases. Currently, commercially available ELISA assays are based on a mixture of O-antigens (LPS or total cell lysate of Salmonella and are hampered by cross-reaction. The identification of novel immunogenic proteins would be useful to develop ELISA based diagnostic assays with a higher specificity. Results A phage display library of the entire Salmonella Typhimurium genome was constructed and 47 immunogenic oligopeptides were identified using a pool of convalescent sera from pigs infected with Salmonella Typhimurium. The corresponding complete genes of seven of the identified oligopeptids were cloned. Five of them were produced in E. coli. The immunogenic character of these antigens was validated with sera from pigs infeced with S. Tyhimurium and control sera from non-infected animals. Finally, human antibody fragments (scFv against these five antigens were selected using antibody phage display and characterised. Conclusion In this work, we identified novel immunogenic proteins of Salmonella Typhimurium and generated antibody fragments against these antigens completely based on phage display. Five immunogenic proteins were validated using a panel of positive and negative sera for prospective applications in diagnostics of Salmonela Typhimurium.

  4. Characterization of salt interferences in second-harmonic generation detection of protein crystals

    Science.gov (United States)

    Closser, R. G.; Gualtieri, E. J.; Newman, J. A.; Simpson, G. J.

    2013-01-01

    Studies were undertaken to assess the merits and limitations of second-harmonic generation (SHG) for the selective detection of protein and polypeptide crystal formation, focusing on the potential for false positives from SHG-active salts present in crystallization media. The SHG activities of salts commonly used in protein crystallization were measured and quantitatively compared with reference samples. Out of 19 salts investigated, six produced significant background SHG and 15 of the 96 wells of a sparse-matrix screen produced SHG upon solvent evaporation. SHG-active salts include phosphates, hydrated sulfates, formates and tartrates, while chlorides, acetates and anhydrous sulfates resulted in no detectable SHG activity. The identified SHG-active salts produced a range of signal intensities spanning nearly three orders of magnitude. However, even the weakest SHG-active salt produced signals that were several orders of magnitude greater than those produced by typical protein crystals. In general, SHG-active salts were identifiable through characteristically strong SHG and negligible two-photon-excited ultraviolet fluorescence (TPE-UVF). Exceptions included trials containing either potassium dihydrogen phosphate or ammonium formate, which produced particularly strong SHG, but with residual weak TPE-UVF signals that could potentially complicate discrimination in crystallization experiments using these precipitants. PMID:24282335

  5. Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.

    Directory of Open Access Journals (Sweden)

    Emmanuel Obishakin

    Full Text Available Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa, costly to produce and require cold chain maintenance, a condition that is difficult to achieve in trypanosomiasis endemic regions, which are mostly rural. Nanobodies are recombinant, heat-stable, small-sized (15 kDa, antigen-specific, single-domain, variable fragments derived from heavy chain-only antibodies in camelids. Because of numerous advantages over classical antibodies, we investigated the use of nanobodies for the targeting of trypanosome-specific antigens and diagnostic potential. An alpaca was immunized using lysates of Trypanosoma evansi. Using phage display and bio-panning techniques, a cross-reactive nanobody (Nb392 targeting all trypanosome species and isolates tested was selected. Imunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry assays were combined to identify the target recognized. Nb392 targets paraflagellar rod protein (PFR1 of T. evansi, T. brucei, T. congolense and T. vivax. Two different RNAi mutants with defective PFR assembly (PFR2RNAi and KIF9BRNAi were used to confirm its specificity. In conclusion, using a complex protein mixture for alpaca immunization, we generated a highly specific nanobody (Nb392 that targets a conserved trypanosome protein, i.e., PFR1 in the flagella of trypanosomes. Nb392 is an excellent marker for the PFR and can be useful in the diagnosis of trypanosomiasis. In addition, as demonstrated, Nb392 can be a useful research or PFR protein isolation tool.

  6. The Presence, Persistence and Functional Properties of Plasmodium vivax Duffy Binding Protein II Antibodies Are Influenced by HLA Class II Allelic Variants

    Science.gov (United States)

    Torres, Leticia M.; Lima, Barbara A. S.; Sousa, Taís N.; Alves, Jéssica R. S.; Rocha, Roberto S.; Fontes, Cor J. F.; Sanchez, Bruno A. M.; Adams, John H.; Brito, Cristiana F. A.; Pires, Douglas E. V.; Ascher, David B.; Sell, Ana Maria; Carvalho, Luzia H.

    2016-01-01

    Background The human malaria parasite Plasmodium vivax infects red blood cells through a key pathway that requires interaction between Duffy binding protein II (DBPII) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). A high proportion of P. vivax-exposed individuals fail to develop antibodies that inhibit DBPII-DARC interaction, and genetic factors that modulate this humoral immune response are poorly characterized. Here, we investigate if DBPII responsiveness could be HLA class II-linked. Methodology/Principal Findings A community-based open cohort study was carried out in an agricultural settlement of the Brazilian Amazon, in which 336 unrelated volunteers were genotyped for HLA class II (DRB1, DQA1 and DQB1 loci), and their DBPII immune responses were monitored over time (baseline, 6 and 12 months) by conventional serology (DBPII IgG ELISA-detected) and functional assays (inhibition of DBPII–erythrocyte binding). The results demonstrated an increased susceptibility of the DRB1*13:01 carriers to develop and sustain an anti-DBPII IgG response, while individuals with the haplotype DRB1*14:02-DQA1*05:03-DQB1*03:01 were persistent non-responders. HLA class II gene polymorphisms also influenced the functional properties of DBPII antibodies (BIAbs, binding inhibitory antibodies), with three alleles (DRB1*07:01, DQA1*02:01 and DQB1*02:02) comprising a single haplotype linked with the presence and persistence of the BIAbs response. Modelling the structural effects of the HLA-DRB1 variants revealed a number of differences in the peptide-binding groove, which is likely to lead to altered antigen binding and presentation profiles, and hence may explain the differences in subject responses. Conclusions/Significance The current study confirms the heritability of the DBPII antibody response, with genetic variation in HLA class II genes influencing both the development and persistence of IgG antibody responses. Cellular studies to increase

  7. The power of multiplexed functional analysis of genetic variants.

    Science.gov (United States)

    Gasperini, Molly; Starita, Lea; Shendure, Jay

    2016-10-01

    New technologies have recently enabled saturation mutagenesis and functional analysis of nearly all possible variants of regulatory elements or proteins of interest in single experiments. Here we discuss the past, present, and future of such multiplexed (functional) assays for variant effects (MAVEs). MAVEs provide detailed insight into sequence-function relationships, and they may prove critical for the prospective clinical interpretation of genetic variants.

  8. Combining classifiers generated by multi-gene genetic programming for protein fold recognition using genetic algorithm.

    Science.gov (United States)

    Bardsiri, Mahshid Khatibi; Eftekhari, Mahdi; Mousavi, Reza

    2015-01-01

    In this study the problem of protein fold recognition, that is a classification task, is solved via a hybrid of evolutionary algorithms namely multi-gene Genetic Programming (GP) and Genetic Algorithm (GA). Our proposed method consists of two main stages and is performed on three datasets taken from the literature. Each dataset contains different feature groups and classes. In the first step, multi-gene GP is used for producing binary classifiers based on various feature groups for each class. Then, different classifiers obtained for each class are combined via weighted voting so that the weights are determined through GA. At the end of the first step, there is a separate binary classifier for each class. In the second stage, the obtained binary classifiers are combined via GA weighting in order to generate the overall classifier. The final obtained classifier is superior to the previous works found in the literature in terms of classification accuracy.

  9. Denatured G-protein coupled receptors as immunogens to generate highly specific antibodies.

    Science.gov (United States)

    Talmont, Franck; Moulédous, Lionel; Boué, Jérôme; Mollereau, Catherine; Dietrich, Gilles

    2012-01-01

    G-protein coupled receptors (GPCRs) play a major role in a number of physiological and pathological processes. Thus, GPCRs have become the most frequent targets for development of new therapeutic drugs. In this context, the availability of highly specific antibodies may be decisive to obtain reliable findings on localization, function and medical relevance of GPCRs. However, the rapid and easy generation of highly selective anti-GPCR antibodies is still a challenge. Herein, we report that highly specific antibodies suitable for detection of GPCRs in native and unfolded forms can be elicited by immunizing animals against purified full length denatured recombinant GPCRs. Contrasting with the currently admitted postulate, our study shows that an active and well-folded GPCR is not required for the production of specific anti-GPCR antibodies. This new immunizing strategy validated with three different human GPCR (μ-opioid, κ-opioid, neuropeptide FF2 receptors) might be generalized to other members of the GPCR family.

  10. Beyond rotamers: a generative, probabilistic model of side chains in proteins

    DEFF Research Database (Denmark)

    Harder, Tim; Boomsma, Wouter; Paluszewski, Martin;

    2010-01-01

    Background: Accurately covering the conformational space of amino acid side chains is essential for important applications such as protein design, docking and high resolution structure prediction. Today, the most common way to capture this conformational space is through rotamer libraries discrete...... for certain applications. For example, rigorously combining rotamers with physical force fields is associated with numerous problems. Results: In this work we present BASILISK: a generative, probabilistic model of the conformational space of side chains that makes it possible to sample in continuous space...... model of side chain conformational space. We also illustrate how the model can be used for rigorous, unbiased sampling with a physical force field, and how it improves side chain prediction when used as a pseudo-energy term. In conclusion, BASILISK is an important step forward on the way to a rigorous...

  11. Replacement of isoleucine-397 by threonine in the clotting proteinase factor IXa (Los Angeles and Long Beach variants) affects macromolecular catalysis but not L-tosylarginine methyl ester hydrolysis. Lack of correlation between the ox brain prothrombin time and the mutation site in the variant proteins.

    Science.gov (United States)

    Spitzer, S G; Warn-Cramer, B J; Kasper, C K; Bajaj, S P

    1990-01-01

    Previously, from the plasma of unrelated haemophilia-B patients, we isolated two non-functional Factor IX variants, namely Los Angeles (IXLA) and Long Beach (IXLB). Both variants could be cleaved to yield Factor IXa-like molecules, but were defective in catalysing the cleavage of Factor X (macromolecular substrate) and in binding to antithrombin III (macromolecular inhibitor). In the present study we have identified the mutation of IXLA by amplifying the exons (including flanking regions) as well as the 5' end of the gene by polymerase-chain-reaction (PCR) method and sequencing the amplified DNA by the dideoxy chain-termination method. Comparison of the normal IX and IXLA sequences revealed only one base substitution (T----C) in exon VIII of IXLA, with a predicted replacement of Ile-397 to Thr in the mature protein. This mutation is the same as found recently for IXLB. The observation that IXLB and IXLA have the same mutation is an unexpected finding, since, on the basis of their ox brain prothrombin time (PT, a test that measures the ability of the variant Factor IX molecules to inhibit the activation of Factor X by Factor VIIa-tissue factor complex), these variants have been classified into two different groups and were thought to be genetically different. Our observation thus suggests that the ox brain PT does not reflect the locus of mutation in the coding region of the variant molecules. However, our analysis suggests that the ox brain PT is related to Factor IX antigen concentration in the patient's plasma. Importantly, although the mutation in IXLA or IXLB protein is in the catalytic domain, purified IXaLA and IXaLB hydrolyse L-tosylarginine methyl ester at rates very similar to that of normal IXa. These data, in conjunction with our recent data on Factor IXBm Lake Elsinore (Ala-390----Val mutant), strengthen a conclusion that the peptide region containing residues 390-397 of normal Factor IXa plays an essential role in macromolecular substrate catalysis and

  12. rMotifGen: random motif generator for DNA and protein sequences

    Directory of Open Access Journals (Sweden)

    Hardin C Timothy

    2007-08-01

    Full Text Available Abstract Background Detection of short, subtle conserved motif regions within a set of related DNA or amino acid sequences can lead to discoveries about important regulatory domains such as transcription factor and DNA binding sites as well as conserved protein domains. In order to help assess motif detection algorithms on motifs with varying properties and levels of conservation, we have developed a computational tool, rMotifGen, with the sole purpose of generating a number of random DNA or protein sequences containing short sequence motifs. Each motif consensus can be user-defined, randomly generated, or created from a position-specific scoring matrix (PSSM. Insertions and mutations within these motifs are created according to user-defined parameters and substitution matrices. The resulting sequences can be helpful in mutational simulations and in testing the limits of motif detection algorithms. Results Two implementations of rMotifGen have been created, one providing a graphical user interface (GUI for random motif construction, and the other serving as a command line interface. The second implementation has the added advantages of platform independence and being able to be called in a batch mode. rMotifGen was used to construct sample sets of sequences containing DNA motifs and amino acid motifs that were then tested against the Gibbs sampler and MEME packages. Conclusion rMotifGen provides an efficient and convenient method for creating random DNA or amino acid sequences with a variable number of motifs, where the instance of each motif can be incorporated using a position-specific scoring matrix (PSSM or by creating an instance mutated from its corresponding consensus using an evolutionary model based on substitution matrices. rMotifGen is freely available at: http://bioinformatics.louisville.edu/brg/rMotifGen/.

  13. Genomic DNA pooling strategy for next-generation sequencing-based rare variant discovery in abdominal aortic aneurysm regions of interest-challenges and limitations

    NARCIS (Netherlands)

    Harakalova, M.; Nijman, I.J.; Medic, J.; Mokry, M.; Renkens, I.; Blankensteijn, J.D.; Kloosterman, W.P.; Baas, A.F.; Cuppen, E.

    2011-01-01

    The costs and efforts for sample preparation of hundreds of individuals, their genomic enrichment for regions of interest, and sufficient deep sequencing bring a significant burden to next-generation sequencing-based experiments. We investigated whether pooling of samples at the level of genomic DNA

  14. A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.

    Science.gov (United States)

    Magnani, Francesca; Serrano-Vega, Maria J; Shibata, Yoko; Abdul-Hussein, Saba; Lebon, Guillaume; Miller-Gallacher, Jennifer; Singhal, Ankita; Strege, Annette; Thomas, Jennifer A; Tate, Christopher G

    2016-08-01

    The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6-12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an (125)I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies.

  15. Bacteriophage epitope libraries. The generation of specific binding proteins and peptides in vitro.

    Science.gov (United States)

    Fink, L M; Hsu, P L

    1994-01-01

    New concepts and methodologies that can be used to generate proteins, such as specific variable regions of immunoglobulins and other binding peptides in an in vitro selection system are reviewed. These technologies can also be used to alter the kinetics, affinity and avidity of various binding interactions. The nature of epitopes recognized by specific antibodies or receptors can be delineated using selected epitopes displayed on bacteriophages. The basic principles of the technology is predicted upon the belief that if one has a large enough variety of keys, one can open any given lock. The range of utility of these systems to generate new reagents will impact upon the development of new diagnostic and therapeutic reagents. This technology should allow for a much wider range of probes which may have increased binding capacity and allow the development of more sensitive assays with higher signal to noise ratios. These reagents can be produced more efficiently without the use of animals and will be used in diagnostic and experimental pathology. This brief review presents a concise description of the concepts and uses of this new technology. Selected references and reviews are given as sources for further details.

  16. Neutrophil-generated oxidative stress and protein damage in Staphylococcus aureus.

    Science.gov (United States)

    Beavers, William N; Skaar, Eric P

    2016-08-01

    Staphylococcus aureus is a ubiquitous, versatile and dangerous pathogen. It colonizes over 30% of the human population, and is one of the leading causes of death by an infectious agent. During S. aureus colonization and invasion, leukocytes are recruited to the site of infection. To combat S. aureus, leukocytes generate an arsenal of reactive species including superoxide, hydrogen peroxide, nitric oxide and hypohalous acids that modify and inactivate cellular macromolecules, resulting in growth defects or death. When S. aureus colonization cannot be cleared by the immune system, antibiotic treatment is necessary and can be effective. Yet, this organism quickly gains resistance to each new antibiotic it encounters. Therefore, it is in the interest of human health to acquire a deeper understanding of how S. aureus evades killing by the immune system. Advances in this field will have implications for the design of future S. aureus treatments that complement and assist the host immune response. In that regard, this review focuses on how S. aureus avoids host-generated oxidative stress, and discusses the mechanisms used by S. aureus to survive oxidative damage including antioxidants, direct repair of damaged proteins, sensing oxidant stress and transcriptional changes. This review will elucidate areas for studies to identify and validate future antimicrobial targets.

  17. Oncogenicity and Selective Inhibition of ERG Splicing Variants in Prostate Cancer

    Science.gov (United States)

    2012-03-01

    promoters and the red circles mark the principal polyA sites. On the bottom, the protein domain structure is schematized with the corresponding...includes variants showing skipping of exons 2, 5, 7 and 8; usage of a proximal (Short) polyA site in exon 11 (11SpA) or of an additional intronic one...exons 4 and 7b) and three main polyA sites (7bpA, 11LpA, 12pA) combine to generate 30 principal mRNA variants, which can encode 15 different ERG

  18. Discovery of the improved antagonistic prolactin variants by library screening.

    Science.gov (United States)

    Liu, Yun; Gong, Wei; Breinholt, Jens; Nørskov-Lauritsen, Leif; Zhang, Jinchao; Ma, Qinhong; Chen, Jianhe; Panina, Svetlana; Guo, Wei; Li, Tengkun; Zhang, Jingyuan; Kong, Meng; Liu, Zibing; Mao, Jingjing; Christensen, Leif; Hu, Sean; Wang, Lingyun

    2011-11-01

    Prolactin (PRL), a potent growth stimulator of the mammary epithelium, has been suggested to be a factor contributing to the development and progression of breast and prostate cancer. Several PRL receptor (PRLR) antagonists have been identified in the past decades, but their in vivo growth inhibitory potency was restricted by low receptor affinity, rendering them pharmacologically unattractive for clinical treatment. Thus, higher receptor affinity is essential for the development of improved PRLR antagonistic variants with improved in vivo potency. In this study, we generated Site 1 focused protein libraries of human G129R-PRL mutants and screened for those with increased affinity to the human PRLR. By combining the mutations with enhanced affinities for PRLR, we identified a novel G129R-PRL variant with mutations at Site 1 that render nearly 50-fold increase in the antagonistic potency in vitro.

  19. Rare variant density across the genome and across populations

    OpenAIRE

    Raska Paola; Zhu Xiaofeng

    2011-01-01

    Abstract Next-generation sequencing allows for a new focus on rare variant density for conducting analyses of association to disease and for narrowing down the genomic regions that show evidence of functionality. In this study we use the 1000 Genomes Project pilot data as distributed by Genetic Analysis Workshop 17 to compare rare variant densities across seven populations. We made the comparisons using regressions of rare variants on total variant counts per gene for each population and Taji...

  20. Detection of ST772 Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus (Bengal Bay clone and ST22 S. aureus isolates with a genetic variant of elastin binding protein in Nepal

    Directory of Open Access Journals (Sweden)

    R.H. Pokhrel

    2016-05-01

    Full Text Available Genetic characteristics were analysed for recent clinical isolates of methicillin-resistant and -susceptible Staphylococcus aureus (MRSA and MSSA respectively in Kathmandu, Nepal. MRSA isolates harbouring Panton-Valentine leukocidin (PVL genes were classified into ST1, ST22 and ST88 with SCCmec-IV and ST772 with SCCmec-V (Bengal Bay clone, while PVL-positive MSSA into ST22, ST30 and ST772. ST22 isolates (PVL-positive MRSA and MSSA, PVL-negative MRSA possessed a variant of elastin binding protein gene (ebpS with an internal deletion of 180 bp, which was similar to that reported for ST121 S. aureus previously outside Nepal. Phylogenetic analysis indicated that the ebpS variant in ST22 might have occurred independently of ST121 strains. This is the first report of ST772 PVL-positive MRSA in Nepal and detection of the deletion variant of ebpS in ST22 S. aureus.

  1. The Effect of Turmeric (Curcuma longa Extract on the Functionality of the Solute Carrier Protein 22 A4 (SLC22A4 and Interleukin-10 (IL-10 Variants Associated with Inflammatory Bowel Disease

    Directory of Open Access Journals (Sweden)

    Mark J. McCann

    2014-10-01

    Full Text Available Inflammatory bowel disease (IBD is a chronic relapsing disease. Genetic predisposition to the disease reduces an individual’s capacity to respond appropriately to environmental challenges in the intestine leading to inappropriate inflammation. IBD patients often modify their diet to mitigate or reduce the severity of inflammation. Turmeric (Curcuma longa L., Zingiberaceae has historically been used in Chinese, Hindu, and Ayurvedic medicine over several centuries to treat inflammatory disorders. To understand how turmeric may influence the consequences of a genetic predisposition to inappropriate inflammation, we used HEK293 cells to examine the in vitro capacity of turmeric extract and fractions to affect the functionality of two gene variants, solute carrier protein 22 A4 (SLC22A4, rs1050152 and interleukin-10 (IL-10, rs1800896 associated with IBD. We found that a turmeric extract and several chromatographically separated fractions beneficially affected the variants of SLC22A4 and IL-10 associated with IBD, by reducing inappropriate epithelial cell transport (SLC22A4, 503F and increasing anti-inflammatory cytokine gene promoter activity (IL-10, −1082A. The effect of turmeric on the IL-10 variant was strongly associated with the curcumin content of the extract and its fractions.

  2. The effect of turmeric (Curcuma longa) extract on the functionality of the solute carrier protein 22 A4 (SLC22A4) and interleukin-10 (IL-10) variants associated with inflammatory bowel disease.

    Science.gov (United States)

    McCann, Mark J; Johnston, Sarah; Reilly, Kerri; Men, Xuejing; Burgess, Elaine J; Perry, Nigel B; Roy, Nicole C

    2014-10-13

    Inflammatory bowel disease (IBD) is a chronic relapsing disease. Genetic predisposition to the disease reduces an individual's capacity to respond appropriately to environmental challenges in the intestine leading to inappropriate inflammation. IBD patients often modify their diet to mitigate or reduce the severity of inflammation. Turmeric (Curcuma longa L., Zingiberaceae) has historically been used in Chinese, Hindu, and Ayurvedic medicine over several centuries to treat inflammatory disorders. To understand how turmeric may influence the consequences of a genetic predisposition to inappropriate inflammation, we used HEK293 cells to examine the in vitro capacity of turmeric extract and fractions to affect the functionality of two gene variants, solute carrier protein 22 A4 (SLC22A4, rs1050152) and interleukin-10 (IL-10, rs1800896) associated with IBD. We found that a turmeric extract and several chromatographically separated fractions beneficially affected the variants of SLC22A4 and IL-10 associated with IBD, by reducing inappropriate epithelial cell transport (SLC22A4, 503F) and increasing anti-inflammatory cytokine gene promoter activity (IL-10, -1082A). The effect of turmeric on the IL-10 variant was strongly associated with the curcumin content of the extract and its fractions.

  3. Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1

    DEFF Research Database (Denmark)

    End, Caroline; Lyer, Stefan; Renner, Marcus

    2005-01-01

    Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant...... yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup...

  4. Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein.

    Science.gov (United States)

    Buvoli, M; Cobianchi, F; Bestagno, M G; Mangiarotti, A; Bassi, M T; Biamonti, G; Riva, S

    1990-01-01

    The human hnRNP core protein A1 (34 kd) is encoded by a 4.6 kb gene split into 10 exons. Here we show that the A1 gene can be differentially spliced by the addition of an extra exon. The new transcript encodes a minor protein of the hnRNP complex, here defined A1B protein, with a calculated mol. wt of 38 kd, that coincides with a protein previously designated as B2 by some authors. In vitro translation of the mRNAs selected by hybridization with A1 cDNA produced two proteins of 34 and 38 kd; Northern blot analysis of poly(A)+ RNA from HeLa cells revealed that the abundance of the A1B mRNA was approximately 5% that of A1. The A1B protein was detected by Western blotting with an anti-A1 monoclonal antibody both in enriched preparations of basic hnRNP proteins and in 40S hnRNP particles. The A1B protein exhibits a significantly higher affinity than A1 for ssDNA. The recombinant A1B protein, expressed in Escherichia coli, shows the same electrophoretic mobility and charge as the cellular one. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1691095

  5. Dexamethasone partially rescues ataxia telangiectasia-mutated (ATM) deficiency in ataxia telangiectasia by promoting a shortened protein variant retaining kinase activity.

    Science.gov (United States)

    Menotta, Michele; Biagiotti, Sara; Bianchi, Marzia; Chessa, Luciana; Magnani, Mauro

    2012-11-30

    Ataxia telangiectasia (AT) is a rare genetic disease, still incurable, resulting from biallelic mutations in the ataxia telangiectasia-mutated (ATM) gene. Recently, short term treatment with glucocorticoid analogues improved neurological symptoms characteristic of this syndrome. Nevertheless, the molecular mechanism involved in glucocorticoid action in AT patients is not yet known. Here we describe, for the first time in mammalian cells, a short direct repeat-mediated noncanonical splicing event induced by dexamethasone, which leads to the skipping of mutations upstream of nucleotide residue 8450 of ATM coding sequence. The resulting transcript provides an alternative ORF translated in a new ATM variant with the complete kinase domain. This miniATM variant was also highlighted in lymphoblastoid cell lines from AT patients and was shown to be likely active. In conclusion, dexamethasone treatment may partly restore ATM activity in ataxia telangiectasia cells by a new molecular mechanism that overcomes most of the mutations so far described within this gene.

  6. An unusual case of an ACTH-secreting macroadenoma with a germline variant in the aryl hydrocarbon receptor-interacting protein (AIP) gene

    DEFF Research Database (Denmark)

    Dinesen, Pia T; Dal, Jakob; Gabrovska, Plamena;

    2015-01-01

    was diagnosed with a large pituitary tumor by magnetic resonance imaging (MRI). His visual fields were intact and he exhibited no features of CD. Owing to an exuberant response to synacthen, an overnight dexamethasone suppression test was performed revealing inadequate suppression of plasma cortisol (419 nmol...... test demonstrated high basal and stimulated cortisol levels; an overnight dexamethasone suppression test showed no suppression (791 nmol/l) and elevated plasma ACTH levels (135 ng/l). A transcranial operation was performed followed by radiotherapy. Two months after radiotherapy, he developed secondary...... growth rather than symptoms of hypersecretion. The particular AIP gene variant identified in our patient is shared by four other reported cases of CD. Future studies are needed to assess whether the reported AIP gene variant is more than just coincidental. LEARNING POINTS: CD is occasionally dominated...

  7. Generation of Recombinant Schmallenberg Virus Nucleocapsid Protein in Yeast and Development of Virus-Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Justas Lazutka

    2014-01-01

    Full Text Available Schmallenberg virus (SBV, discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs. Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.

  8. Adaptor protein complexes 1 and 3 are essential for generation of synaptic vesicles from activity-dependent bulk endosomes.

    Science.gov (United States)

    Cheung, Giselle; Cousin, Michael A

    2012-04-25

    Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endo