Science.gov

Sample records for chimeric cyanovirin-n homolog

  1. A designed chimeric cyanovirin-N homolog lectin: structure and molecular basis of sucrose binding.

    PubMed

    Koharudin, Leonardus M I; Furey, William; Gronenborn, Angela M

    2009-12-01

    The NMR and X-ray structures of a designed chimeric cyanovirin-N homolog (CVNH) protein were determined. The individual halves of the structure are similar to their counterparts in the parent proteins, with domains A and B resembling the structures of TbCVNH and NcCVNH, respectively. No significant differences between the solution and crystal conformations were observed, although details in loop conformations and distinct crystal packing-induced features are present. Carbohydrate binding studies by NMR revealed affinity and specificity for Glc alpha(1-2)Frc and Man alpha(1-2)Man, and the parental half that is devoid of any sucrose affinity in NcCVNH was transformed into a genuine sucrose binding site in the context of the chimera. The atomic details of sugar recognition are seen in the crystal structure of the protein with two bound Glc alpha(1-2)Frc molecules. Both sugars exhibit different conformations around the glycosidic bond and engage in unique hydrogen bonding networks in the two sites. Although the protein is able to bind two Man alpha(1-2)Man molecules, a property associated with HIV-inactivation, no anti-HIV activity was observed for the hybrid protein. These results provide the structural basis for sugar recognition in the CVNH family and aid in deciphering the relationship between sugar binding and anti-HIV activity. 2009 Wiley-Liss, Inc.

  2. Carbohydrate recognition by the antiviral lectin cyanovirin-N

    PubMed Central

    Fujimoto, Yukiji K.; Green, David F.

    2012-01-01

    Cyanovirin-N is a cyanobacterial lectin with potent antiviral activity, and has been the focus of extensive pre-clinical investigation as a potential prophylactic for the prevention of the sexual transmission of the human immunodeficiency virus (HIV). Here we present a detailed analysis of carbohydrate recognition by this important protein, using a combination of computational methods, including extensive molecular dynamics simulations and Molecular-Mechanics/ Poisson–Boltzmann/Surface-Area (MM/PBSA) energetic analysis. The simulation results strongly suggest that the observed tendency of wildtype CVN to form domain-swapped dimers is the result of a previously unidentified cis-peptide bond present in the monomeric state. The energetic analysis additionally indicates that the highest-affinity ligand for CVN characterized to date (α-Man-(1,2)-α-Man-(1,2)-α-Man) is recognized asymmetrically by the two binding sites. Finally, we are able to provide a detailed map of the role of all binding site functional groups (both backbone and side chain) to various aspects of molecular recognition: general affinity for cognate ligands, specificity for distinct oligosaccharide targets and the asymmetric recognition of α-Man-(1,2)-α-Man-(1,2)-α-Man. Taken as a whole, these results complement past experimental characterization (both structural and thermodynamic) to provide the most complete understanding of carbohydrate recognition by CVN to date. The results also provide strong support for the application of similar approaches to the understanding of other protein–carbohydrate complexes. PMID:23057413

  3. Potent anti-influenza activity of cyanovirin-N and interactions with viral hemagglutinin.

    PubMed

    O'Keefe, Barry R; Smee, Donald F; Turpin, Jim A; Saucedo, Carrie J; Gustafson, Kirk R; Mori, Toshiyuki; Blakeslee, Dennis; Buckheit, Robert; Boyd, Michael R

    2003-08-01

    The novel antiviral protein cyanovirin-N (CV-N) was initially discovered based on its potent activity against the human immunodeficiency virus (HIV). Subsequent studies identified the HIV envelope glycoproteins gp120 and gp41 as molecular targets of CV-N. More recently, mechanistic studies have shown that certain high-mannose oligosaccharides (oligomannose-8 and oligomannose-9) found on the HIV envelope glycoproteins comprise the specific sites to which CV-N binds. Such selective, carbohydrate-dependent interactions may account, at least in part, for the unusual and unexpected spectrum of antiviral activity of CV-N described herein. We screened CV-N against a broad range of respiratory and enteric viruses, as well as flaviviruses and herpesviruses. CV-N was inactive against rhinoviruses, human parainfluenza virus, respiratory syncytial virus, and enteric viruses but was moderately active against some herpesvirus and hepatitis virus (bovine viral diarrhea virus) strains (50% effective concentration [EC(50)] = approximately 1 micro g/ml) while inactive against others. Remarkably, however, CV-N and related homologs showed highly potent antiviral activity against almost all strains of influenza A and B virus, including clinical isolates and a neuraminidase inhibitor-resistant strain (EC(50) = 0.004 to 0.04 micro g/ml). When influenza virus particles were pretreated with CV-N, viral titers were lowered significantly (>1,000-fold). Further studies identified influenza virus hemagglutinin as a target for CV-N, showed that antiviral activity and hemagglutinin binding were correlated, and indicated that CV-N's interactions with hemagglutinin involved oligosaccharides. These results further reveal new potential avenues for antiviral therapeutics and prophylaxis targeting specific oligosaccharide-comprised sites on certain enveloped viruses, including HIV, influenza virus, and possibly others.

  4. Chimeric mitochondrial minichromosomes of the human body louse, Pediculus humanus: evidence for homologous and non-homologous recombination.

    PubMed

    Shao, Renfu; Barker, Stephen C

    2011-02-15

    The mitochondrial (mt) genome of the human body louse, Pediculus humanus, consists of 18 minichromosomes. Each minichromosome is 3 to 4 kb long and has 1 to 3 genes. There is unequivocal evidence for recombination between different mt minichromosomes in P. humanus. It is not known, however, how these minichromosomes recombine. Here, we report the discovery of eight chimeric mt minichromosomes in P. humanus. We classify these chimeric mt minichromosomes into two groups: Group I and Group II. Group I chimeric minichromosomes contain parts of two different protein-coding genes that are from different minichromosomes. The two parts of protein-coding genes in each Group I chimeric minichromosome are joined at a microhomologous nucleotide sequence; microhomologous nucleotide sequences are hallmarks of non-homologous recombination. Group II chimeric minichromosomes contain all of the genes and the non-coding regions of two different minichromosomes. The conserved sequence blocks in the non-coding regions of Group II chimeric minichromosomes resemble the "recombination repeats" in the non-coding regions of the mt genomes of higher plants. These repeats are essential to homologous recombination in higher plants. Our analyses of the nucleotide sequences of chimeric mt minichromosomes indicate both homologous and non-homologous recombination between minichromosomes in the mitochondria of the human body louse. Copyright © 2010 Elsevier B.V. All rights reserved.

  5. The antiviral lectin cyanovirin-N: probing multivalency and glycan recognition through experimental and computational approaches.

    PubMed

    Woodrum, Brian W; Maxwell, Jason D; Bolia, Ashini; Ozkan, S Banu; Ghirlanda, Giovanna

    2013-10-01

    CVN (cyanovirin-N), a small lectin isolated from cyanobacteria, exemplifies a novel class of anti-HIV agents that act by binding to the highly glycosylated envelope protein gp120 (glycoprotein 120), resulting in inhibition of the crucial viral entry step. In the present review, we summarize recent work in our laboratory and others towards determining the crucial role of multivalency in the antiviral activity, and we discuss features that contribute to the high specificity and affinity for the glycan ligand observed in CVN. An integrated approach that encompasses structural determination, mutagenesis analysis and computational work holds particular promise to clarify aspects of the interactions between CVN and glycans.

  6. Expression, purification, and characterization of recombinant cyanovirin-N for vaginal anti-HIV microbicide development.

    PubMed

    Colleluori, Diana M; Tien, Deborah; Kang, Feirong; Pagliei, Tara; Kuss, Ryan; McCormick, Timothy; Watson, Karen; McFadden, Karyn; Chaiken, Irwin; Buckheit, Robert W; Romano, Joseph W

    2005-02-01

    Cyanovirin-N (CV-N) is a prokaryotic protein under development as a topical anti-HIV microbicide, an urgent and necessary approach to prevent HIV transmission in at-risk populations worldwide. We have expressed recombinant CV-N as inclusion bodies in the cytoplasm of Escherichia coli. A purification scheme has been developed that exploits the physicochemical properties of this protein, in particular its stability in a harsh inclusion body purification scheme. Under the conditions developed, this system yields 140 mg of highly purified CV-N per liter of high-density cell culture, which represents a 14-fold increase over the best recombinant CV-N yield reported to date. This purification scheme results in monomeric CV-N as analyzed by SDS-PAGE, isoelectric focusing, and reverse phase- and size exclusion-HPLC. This recombinantly expressed and refolded CV-N binds to gp120 with nanomolar affinity and retains its potent anti-HIV activities in cell-based assays. The expression and purification system described herein provides a better means for the mass production of CV-N for further microbicide development.

  7. Rhizosecretion improves the production of Cyanovirin-N in Nicotiana tabacum through simplified downstream processing.

    PubMed

    Madeira, Luisa M; Szeto, Tim H; Ma, Julian K-C; Drake, Pascal M W

    2016-07-01

    Rhizosecretion has many advantages for the production of recombinant pharmaceuticals, notably facile downstream processing from hydroponic medium. The aim of this study was to increase yields of the HIV microbicide candidate, Cyanovirin-N (CV-N), obtained using this production platform and to develop a simplified methodology for its downstream processing from hydroponic medium. Placing hydroponic cultures on an orbital shaker more than doubled the concentration of CV-N in the hydroponic medium compared to plants which remained stationary, reaching a maximum of approximately 20μg/ml in one week, which is more than 3 times higher than previously reported yields. The protein composition of the hydroponic medium, the rhizosecretome, was characterised in plants cultured with or without the plant growth regulator alpha-napthaleneacetic acid by LC-ESI-MS/MS, and CV-N was the most abundant protein. The issue of large volumes in the rhizosecretion system was addressed by using ion exchange chromatography to concentrate CV-N and partially remove impurities. The semi-purified CV-N was demonstrated to bind to HIV gp120 in an ELISA and to neutralise HIVBa-L with an IC50 of 6nM in a cell-based assay. Rhizosecretion is therefore a practicable and inexpensive method for the production of functional CV-N.

  8. Rhizosecretion improves the production of Cyanovirin-N in Nicotiana tabacum through simplified downstream processing

    PubMed Central

    Madeira, Luisa M.; Szeto, Tim H.

    2016-01-01

    Rhizosecretion has many advantages for the production of recombinant pharmaceuticals, notably facile downstream processing from hydroponic medium. The aim of this study was to increase yields of the HIV microbicide candidate, Cyanovirin-N (CV-N), obtained using this production platform and to develop a simplified methodology for its downstream processing from hydroponic medium. Placing hydroponic cultures on an orbital shaker more than doubled the concentration of CV-N in the hydroponic medium compared to plants which remained stationary, reaching a maximum of approximately 20μg/ml in one week, which is more than 3 times higher than previously reported yields. The protein composition of the hydroponic medium, the rhizosecretome, was characterised in plants cultured with or without the plant growth regulator alpha-napthaleneacetic acid by LC-ESI-MS/MS, and CV-N was the most abundant protein. The issue of large volumes in the rhizosecretion system was addressed by using ion exchange chromatography to concentrate CV-N and partially remove impurities. The semi-purified CV-N was demonstrated to bind to HIV gp120 in an ELISA and to neutralise HIVBa-L with an IC50 of 6nM in a cell-based assay. Rhizosecretion is therefore a practicable and inexpensive method for the production of functional CV-N. PMID:26901579

  9. The antiviral protein cyanovirin-N: the current state of its production and applications.

    PubMed

    Xiong, Sheng; Fan, Jun; Kitazato, Kaio

    2010-04-01

    Human immunodeficiency virus (HIV)/AIDS continues to spread worldwide, and most of the HIV-infected people living in developing countries have little or no access to highly active antiretroviral therapy. The development of efficient and low-cost microbicides to prevent sexual transmission of HIV should be given high priority because there is no vaccine available yet. Cyanovirin-N (CVN) is an entry inhibitor of HIV and many other viruses, and it represents a new generation of microbicide that has specific and potent activity, a different mechanism of action, and unusual chemicophysical stability. In vitro and in vivo antiviral tests suggested that the anti-HIV effect of CVN is stronger than a well-known gp120-targeted antibody (2G12) and another microbicide candidate, PRO2000. CVN is a cyanobacteria-derived protein that has special structural features, making the artificial production of this protein very difficult. In order to develop an efficient and relatively low-cost approach for large-scale production of recombinant CVN to satisfy medical use, this protein has been expressed in many systems by trial and error. Here, to summarize the potential and remaining challenges for the development of this protein into an HIV prevention agent, the progress in the structural mechanism determination, heterologous production and pharmacological evaluation of CVN is reviewed.

  10. The Antiretroviral Lectin Cyanovirin-N Targets Well-Known and Novel Targets on the Surface of Entamoeba histolytica Trophozoites ▿ †

    PubMed Central

    Carpentieri, Andrea; Ratner, Daniel M.; Ghosh, Sudip K.; Banerjee, Sulagna; Bushkin, G. Guy; Cui, Jike; Lubrano, Michael; Steffen, Martin; Costello, Catherine E.; O'Keefe, Barry; Robbins, Phillips W.; Samuelson, John

    2010-01-01

    Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, has a truncated Asn-linked glycan (N-glycan) precursor composed of seven sugars (Man5GlcNAc2). Here, we show that glycoproteins with unmodified N-glycans are aggregated and capped on the surface of E. histolytica trophozoites by the antiretroviral lectin cyanovirin-N and then replenished from large intracellular pools. Cyanovirin-N cocaps the Gal/GalNAc adherence lectin, as well as glycoproteins containing O-phosphodiester-linked glycans recognized by an anti-proteophosphoglycan monoclonal antibody. Cyanovirin-N inhibits phagocytosis by E. histolytica trophozoites of mucin-coated beads, a surrogate assay for amebic virulence. For technical reasons, we used the plant lectin concanavalin A rather than cyanovirin-N to enrich secreted and membrane proteins for mass spectrometric identification. E. histolytica glycoproteins with occupied N-glycan sites include Gal/GalNAc lectins, proteases, and 17 previously hypothetical proteins. The latter glycoproteins, as well as 50 previously hypothetical proteins enriched by concanavalin A, may be vaccine targets as they are abundant and unique. In summary, the antiretroviral lectin cyanovirin-N binds to well-known and novel targets on the surface of E. histolytica that are rapidly replenished from large intracellular pools. PMID:20852023

  11. A Designed “Nested” Dimer of Cyanovirin-N Increases Antiviral Activity

    PubMed Central

    Woodrum, Brian W.; Maxwell, Jason; Allen, Denysia M.; Wilson, Jennifer; Krumpe, Lauren R.H.; Bobkov, Andrey A.; Hill, R. Blake; Kibler, Karen V.; O’Keefe, Barry R.; Ghirlanda, Giovanna

    2016-01-01

    Cyanovirin-N (CV-N) is an antiviral lectin with potent activity against enveloped viruses, including HIV. The mechanism of action involves high affinity binding to mannose-rich glycans that decorate the surface of enveloped viruses. In the case of HIV, antiviral activity of CV-N is postulated to require multivalent interactions with envelope protein gp120, achieved through a pseudo-repeat of sequence that adopts two near-identical glycan-binding sites, and possibly involves a 3D-domain-swapped dimeric form of CV-N. Here, we present a covalent dimer of CV-N that increases the number of active glycan-binding sites, and we characterize its ability to recognize four glycans in solution. A CV-N variant was designed in which two native repeats were separated by the “nested” covalent insertion of two additional repeats of CV-N, resulting in four possible glycan-binding sites. The resulting Nested CV-N folds into a wild-type-like structure as assessed by circular dichroism and NMR spectroscopy, and displays high thermal stability with a Tm of 59 °C, identical to WT. All four glycan-binding domains encompassed by the sequence are functional as demonstrated by isothermal titration calorimetry, which revealed two sets of binding events to dimannose with dissociation constants Kd of 25 μM and 900 μM, assigned to domains B and B’ and domains A and A’ respectively. Nested CV-N displays a slight increase in activity when compared to WT CV-N in both an anti-HIV cellular assay and a fusion assay. This construct conserves the original binding specifityies of domain A and B, thus indicating correct fold of the two CV-N repeats. Thus, rational design can be used to increase multivalency in antiviral lectins in a controlled manner. PMID:27275831

  12. A Designed "Nested" Dimer of Cyanovirin-N Increases Antiviral Activity.

    PubMed

    Woodrum, Brian W; Maxwell, Jason; Allen, Denysia M; Wilson, Jennifer; Krumpe, Lauren R H; Bobkov, Andrey A; Hill, R Blake; Kibler, Karen V; O'Keefe, Barry R; Ghirlanda, Giovanna

    2016-06-06

    Cyanovirin-N (CV-N) is an antiviral lectin with potent activity against enveloped viruses, including HIV. The mechanism of action involves high affinity binding to mannose-rich glycans that decorate the surface of enveloped viruses. In the case of HIV, antiviral activity of CV-N is postulated to require multivalent interactions with envelope protein gp120, achieved through a pseudo-repeat of sequence that adopts two near-identical glycan-binding sites, and possibly involves a 3D-domain-swapped dimeric form of CV-N. Here, we present a covalent dimer of CV-N that increases the number of active glycan-binding sites, and we characterize its ability to recognize four glycans in solution. A CV-N variant was designed in which two native repeats were separated by the "nested" covalent insertion of two additional repeats of CV-N, resulting in four possible glycan-binding sites. The resulting Nested CV-N folds into a wild-type-like structure as assessed by circular dichroism and NMR spectroscopy, and displays high thermal stability with a Tm of 59 °C, identical to WT. All four glycan-binding domains encompassed by the sequence are functional as demonstrated by isothermal titration calorimetry, which revealed two sets of binding events to dimannose with dissociation constants Kd of 25 μM and 900 μM, assigned to domains B and B' and domains A and A' respectively. Nested CV-N displays a slight increase in activity when compared to WT CV-N in both an anti-HIV cellular assay and a fusion assay. This construct conserves the original binding specifityies of domain A and B, thus indicating correct fold of the two CV-N repeats. Thus, rational design can be used to increase multivalency in antiviral lectins in a controlled manner.

  13. Differential Inhibitory Effects of Cyanovirin-N, Griffithsin, and Scytovirin on Entry Mediated by Envelopes of Gammaretroviruses and Deltaretroviruses

    PubMed Central

    Jensen, Stig M. R.; Ruscetti, Francis W.; Rein, Alan; Bertolette, Daniel C.; Saucedo, Carrie J.; O'Keefe, Barry R.

    2014-01-01

    The antiviral lectins griffithsin (GRFT), cyanovirin-N (CV-N), and scytovirin (SVN), which inhibit several enveloped viruses, including lentiviruses, were examined for their ability to inhibit entry mediated by Env proteins of delta- and gammaretroviruses. The glycoproteins from human T-cell leukemia virus type 1 (HTLV-1) were resistant to the antiviral effects of all three lectins. For gammaretroviruses, CV-N inhibited entry mediated by some but not all of the envelopes examined, whereas GRFT and SVN displayed only little or no effect. PMID:24284326

  14. Phase system selection with fractional factorial design for purification of recombinant cyanovirin-N from a hydroponic culture medium using centrifugal partition chromatography.

    PubMed

    Grudzień, Łukasz; Madeira, Luisa; Fisher, Derek; Ma, Julian; Garrard, Ian

    2013-04-12

    Centrifugal partition chromatography (CPC) with an aqueous two-phase system (ATPS) was used to purify recombinant cyanovirin-N (CV-N) from other proteins which were co-secreted into a hydroponic plant medium in a rhizosecretion process. To achieve satisfactory protein concentration, the purification was preceded by ultrafiltration performed on a 5 kDa filter. ATPS, because of their gentle nature, were selected as the phase system for CPC. A systematic phase system selection was applied. This involved studying the effect of seven parameters of ATPS: polymer type, salt type, the polymer and salt concentration, the polymer molecular weight, pH, and presence of two additional salts; NaCl and NaClO4, which all together gave 320 combinations. design of experiment (DoE) software allowed the reduction of this number to 46. Having tested partitioning of cyanovirin-N and impurities in 46 ATPS, the three best potential phase systems generated by the programme were then tested on the CPC. Out of these three, 13/13% PEG4000 sodium phosphate, pH 3.0, proved to be most effective phase system in the purification of cyanovirin-N, judged by ELISA and SDS-PAGE analysis, as it eliminated most of the impurities from the final cyanovirin-N preparation.

  15. Solution and crystal structures of a sugar binding site mutant of Cyanovirin-N: no evidence of domain-swapping

    PubMed Central

    Matei, Elena; Furey, William; Gronenborn, Angela M.

    2008-01-01

    SUMMARY The cyanobacterial lectin Cyanovirin-N (CV-N) exhibits antiviral activity against HIV at a low nanomolar concentration by interacting with high-mannose oligosaccharides on the virus surface envelope glycoprotein gp120. Atomic structures of wild-type CV-N revealed a monomer in solution and a domain-swapped dimer in the crystal, with the monomer comprising two independent carbohydrate binding sites that individually bind with micromolar affinity to di- and trimannoses. In the mutant CVNmutDB, the binding site on domain B was abolished and the protein was found to be completely inactive against HIV. We determined the solution NMR and crystal structures of this variant and characterized its sugar binding properties. PMID:18682220

  16. Engineering soya bean seeds as a scalable platform to produce cyanovirin-N, a non-ARV microbicide against HIV.

    PubMed

    O'Keefe, Barry R; Murad, André M; Vianna, Giovanni R; Ramessar, Koreen; Saucedo, Carrie J; Wilson, Jennifer; Buckheit, Karen W; da Cunha, Nicolau B; Araújo, Ana Claudia G; Lacorte, Cristiano C; Madeira, Luisa; McMahon, James B; Rech, Elibio L

    2015-09-01

    There is an urgent need to provide effective anti-HIV microbicides to resource-poor areas worldwide. Some of the most promising microbicide candidates are biotherapeutics targeting viral entry. To provide biotherapeutics to poorer areas, it is vital to reduce the cost. Here, we report the production of biologically active recombinant cyanovirin-N (rCV-N), an antiviral protein, in genetically engineered soya bean seeds. Pure, biologically active rCV-N was isolated with a yield of 350 μg/g of dry seed weight. The observed amino acid sequence of rCV-N matched the expected sequence of native CV-N, as did the mass of rCV-N (11 009 Da). Purified rCV-N from soya is active in anti-HIV assays with an EC50 of 0.82-2.7 nM (compared to 0.45-1.8 nM for E. coli-produced CV-N). Standard industrial processing of soya bean seeds to harvest soya bean oil does not diminish the antiviral activity of recovered rCV-N, allowing the use of industrial soya bean processing to generate both soya bean oil and a recombinant protein for anti-HIV microbicide development. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  17. Linker-extended native cyanovirin-N facilitates PEGylation and potently inhibits HIV-1 by targeting the glycan ligand.

    PubMed

    Chen, Jia; Huang, Dane; Chen, Wei; Guo, Chaowan; Wei, Bo; Wu, Chongchao; Peng, Zhou; Fan, Jun; Hou, Zhibo; Fang, Yongsheng; Wang, Yifei; Kitazato, Kaio; Yu, Guoying; Zou, Chunbin; Qian, Chuiwen; Xiong, Sheng

    2014-01-01

    Cyanovirin-N (CVN) potently inhibits human immunodeficiency virus type 1 (HIV-1) infection, but both cytotoxicity and immunogenicity have hindered the translation of this protein into a viable therapeutic. A molecular docking analysis suggested that up to 12 residues were involved in the interaction of the reverse parallel CVN dimer with the oligosaccharide targets, among which Leu-1 was the most prominent hot spot residue. This finding provided a possible explanation for the lack of anti-HIV-1 activity observed with N-terminal PEGylated CVN. Therefore, linker-CVN (LCVN) was designed as a CVN derivative with a flexible and hydrophilic linker (Gly4Ser)3 at the N-terminus. The N-terminal α-amine of LCVN was PEGylated to create 10 K PEG-aldehyde (ALD)-LCVN. LCVN and 10 K PEG-ALD-LCVN retained the specificity and affinity of CVN for high mannose N-glycans. Moreover, LCVN exhibited significant anti-HIV-1 activity with attenuated cytotoxicity in the HaCaT keratinocyte cell line and MT-4 T lymphocyte cell lines. 10 K PEG-ALD-LCVN also efficiently inactivated HIV-1 with remarkably decreased cytotoxicity and pronounced cell-to-cell fusion inhibitory activity in vitro. The linker-extended CVN and the mono-PEGylated derivative were determined to be promising candidates for the development of an anti-HIV-1 agent. This derivatization approach provided a model for the PEGylation of biologic candidates without introducing point mutations.

  18. Rational and Computational Design of Stabilized Variants of Cyanovirin-N which Retain Affinity and Specificity for Glycan Ligands

    PubMed Central

    Patsalo, Vadim; Raleigh, Daniel P.; Green, David F.

    2011-01-01

    Cyanovirin-N (CVN) is an 11-kDa pseudo-symmetric cyanobacterial lectin that has been shown to inhibit infection by the Human Immunodeficiency Virus (HIV) by binding to high-mannose oligosaccharides on the surface of the viral envelope glycoprotein gp120. In this work we describe rationally-designed CVN variants that stabilize the protein fold while maintaining high affinity and selectivity for their glycan targets. Poisson–Boltzmann calculations and protein repacking algorithms were used to select stabilizing mutations in the protein core. By substituting the buried polar side chains of Ser11, Ser20, and Thr61 with aliphatic groups, we stabilized CVN by nearly 12 °C against thermal denaturation, and by 1 m of GuaHCl against chemical denaturation, relative to a previously-characterized stabilized mutant. Glycan microarray binding experiments confirmed that the specificity profile of carbohydrate binding is unperturbed by the mutations, and is identical for all variants. In particular, the variants selectively bound glycans containing the Manα(1→2)Man linkage, which is the known minimal binding unit of CVN. We also report the slow denaturation kinetics of CVN and show that they can complicate thermodynamic analysis; in particular, the unfolding of CVN cannot be described as a fixed two-state transition. Accurate thermodynamic parameters are needed to describe the complicated free energy landscape of CVN, and we provide updated values for CVN unfolding. PMID:22032696

  19. Solution and crystal structures of a sugar binding site mutant of cyanovirin-N: no evidence of domain swapping.

    PubMed

    Matei, Elena; Furey, William; Gronenborn, Angela M

    2008-08-06

    The cyanobacterial lectin Cyanovirin-N (CV-N) exhibits antiviral activity against HIV at a low nanomolar concentration by interacting with high-mannose oligosaccharides on the virus surface envelope glycoprotein gp120. Atomic structures of wild-type CV-N revealed a monomer in solution and a domain-swapped dimer in the crystal, with the monomer comprising two independent carbohydrate binding sites that individually bind with micromolar affinity to di- and trimannoses. In the mutant CVN(mutDB), the binding site on domain B was abolished and the protein was found to be completely inactive against HIV. We determined the solution NMR and crystal structures of this variant and characterized its sugar binding properties. In solution and the crystal, CVN(mutDB) is a monomer and no domain-swapping was observed. The protein binds to Man-3 and Man-9 with similar dissociation constants ( approximately 4 muM). This confirms that the nanomolar activity of wild-type CV-N is related to the multisite nature of the protein carbohydrate interaction.

  20. Hypothesis: Artifacts, Including Spurious Chimeric RNAs with a Short Homologous Sequence, Caused by Consecutive Reverse Transcriptions and Endogenous Random Primers

    PubMed Central

    Peng, Zhiyu; Yuan, Chengfu; Zellmer, Lucas; Liu, Siqi; Xu, Ningzhi; Liao, D. Joshua

    2015-01-01

    Recent RNA-sequencing technology and associated bioinformatics have led to identification of tens of thousands of putative human chimeric RNAs, i.e. RNAs containing sequences from two different genes, most of which are derived from neighboring genes on the same chromosome. In this essay, we redefine “two neighboring genes” as those producing individual transcripts, and point out two known mechanisms for chimeric RNA formation, i.e. transcription from a fusion gene or trans-splicing of two RNAs. By our definition, most putative RNA chimeras derived from canonically-defined neighboring genes may either be technical artifacts or be cis-splicing products of 5'- or 3'-extended RNA of either partner that is redefined herein as an unannotated gene, whereas trans-splicing events are rare in human cells. Therefore, most authentic chimeric RNAs result from fusion genes, about 1,000 of which have been identified hitherto. We propose a hypothesis of “consecutive reverse transcriptions (RTs)”, i.e. another RT reaction following the previous one, for how most spurious chimeric RNAs, especially those containing a short homologous sequence, may be generated during RT, especially in RNA-sequencing wherein RNAs are fragmented. We also point out that RNA samples contain numerous RNA and DNA shreds that can serve as endogenous random primers for RT and ensuing polymerase chain reactions (PCR), creating artifacts in RT-PCR. PMID:26000048

  1. Hypothesis: Artifacts, Including Spurious Chimeric RNAs with a Short Homologous Sequence, Caused by Consecutive Reverse Transcriptions and Endogenous Random Primers.

    PubMed

    Peng, Zhiyu; Yuan, Chengfu; Zellmer, Lucas; Liu, Siqi; Xu, Ningzhi; Liao, D Joshua

    2015-01-01

    Recent RNA-sequencing technology and associated bioinformatics have led to identification of tens of thousands of putative human chimeric RNAs, i.e. RNAs containing sequences from two different genes, most of which are derived from neighboring genes on the same chromosome. In this essay, we redefine "two neighboring genes" as those producing individual transcripts, and point out two known mechanisms for chimeric RNA formation, i.e. transcription from a fusion gene or trans-splicing of two RNAs. By our definition, most putative RNA chimeras derived from canonically-defined neighboring genes may either be technical artifacts or be cis-splicing products of 5'- or 3'-extended RNA of either partner that is redefined herein as an unannotated gene, whereas trans-splicing events are rare in human cells. Therefore, most authentic chimeric RNAs result from fusion genes, about 1,000 of which have been identified hitherto. We propose a hypothesis of "consecutive reverse transcriptions (RTs)", i.e. another RT reaction following the previous one, for how most spurious chimeric RNAs, especially those containing a short homologous sequence, may be generated during RT, especially in RNA-sequencing wherein RNAs are fragmented. We also point out that RNA samples contain numerous RNA and DNA shreds that can serve as endogenous random primers for RT and ensuing polymerase chain reactions (PCR), creating artifacts in RT-PCR.

  2. Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones.

    PubMed

    Arenhart, Sandra; Silva, José Valter Joaquim; Flores, Eduardo Furtado; Weiblen, Rudi; Gil, Laura Helena Vega Gonzales

    The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV) strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3). The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  3. Effect of the Crystal Environment on Side-Chain Conformational Dynamics in Cyanovirin-N Investigated through Crystal and Solution Molecular Dynamics Simulations

    PubMed Central

    Ahlstrom, Logan S.; Vorontsov, Ivan I.; Shi, Jun; Miyashita, Osamu

    2017-01-01

    Side chains in protein crystal structures are essential for understanding biochemical processes such as catalysis and molecular recognition. However, crystal packing could influence side-chain conformation and dynamics, thus complicating functional interpretations of available experimental structures. Here we investigate the effect of crystal packing on side-chain conformational dynamics with crystal and solution molecular dynamics simulations using Cyanovirin-N as a model system. Side-chain ensembles for solvent-exposed residues obtained from simulation largely reflect the conformations observed in the X-ray structure. This agreement is most striking for crystal-contacting residues during crystal simulation. Given the high level of correspondence between our simulations and the X-ray data, we compare side-chain ensembles in solution and crystal simulations. We observe large decreases in conformational entropy in the crystal for several long, polar and contacting residues on the protein surface. Such cases agree well with the average loss in conformational entropy per residue upon protein folding and are accompanied by a change in side-chain conformation. This finding supports the application of surface engineering to facilitate crystallization. Our simulation-based approach demonstrated here with Cyanovirin-N establishes a framework for quantitatively comparing side-chain ensembles in solution and in the crystal across a larger set of proteins to elucidate the effect of the crystal environment on protein conformations. PMID:28107510

  4. Nonallelic homologous recombination of the FCGR2/3 locus results in copy number variation and novel chimeric FCGR2 genes with aberrant functional expression.

    PubMed

    Nagelkerke, S Q; Tacke, C E; Breunis, W B; Geissler, J; Sins, J W R; Appelhof, B; van den Berg, T K; de Boer, M; Kuijpers, T W

    2015-09-01

    The human FCGR2/3 locus, containing five highly homologous genes encoding the major IgG receptors, shows extensive copy number variation (CNV) associated with susceptibility to autoimmune diseases. Having genotyped >4000 individuals, we show that all CNV at this locus can be explained by nonallelic homologous recombination (NAHR) of the two paralogous repeats that constitute the majority of the locus, and describe four distinct CNV regions (CNRs) with a highly variable prevalence in the population. Apart from CNV, NAHR events also created several hitherto unidentified chimeric FCGR2 genes. These include an FCGR2A/2C chimeric gene that causes a decreased expression of FcγRIIa on phagocytes, resulting in a decreased production of reactive oxygen species in response to immune complexes, compared with wild-type FCGR2A. Conversely, FCGR2C/2A chimeric genes were identified to lead to an increased expression of FCGR2C. Finally, a rare FCGR2B null-variant allele was found, in which a polymorphic stop codon of FCGR2C is introduced into one FCGR2B gene, resulting in a 50% reduction in protein expression. Our study on CNRs and the chimeric genes is essential for the correct interpretation of association studies on FCGR genes as a determinant for disease susceptibility, and may explain some as yet unidentified extreme phenotypes of immune-mediated disease.

  5. The lectins Griffithsin, Cyanovirin-N and Scytovirin inhibit HIV-1 binding to the DC-SIGN receptor and transfer to CD4+ cells

    PubMed Central

    Alexandre, Kabamba B.; Gray, Elin S.; Mufhandu, Hazel; McMahon, James B.; Chakauya, Ereck; O’Keefe, Barry R.; Chikwamba, Rachel; Morris, Lynn

    2011-01-01

    It is generally believed that during the sexual transmission of HIV-1, the glycan-specific DC-SIGN receptor binds the virus and mediates its transfer to CD4+ cells. The lectins griffithsin (GRFT), cyanovirin-N (CV-N) and scytovirin (SVN) inhibit HIV-1 infection by binding to mannose-rich glycans on gp120. We measured the ability of these lectins to inhibit both the HIV-1 binding to DC-SIGN and the DC-SIGN-mediated HIV-1 infection of CD4+ cells. While GRFT, CV-N and SVN were moderately inhibitory to DC-SIGN binding, they potently inhibited DC-SIGN-transfer of HIV-1. The introduction of the 234 glycosylation site abolished HIV-1 sensitivity to lectin inhibition of binding to DC-SIGN and virus transfer to susceptible cells. However, the addition of the 295 glycosylation site increased the inhibition of transfer. Our data suggest that GRFT, CV-N and SVN can block two important stages of the sexual transmission of HIV-1, DC-SIGN binding and transfer, supporting their further development as microbicides. PMID:22209231

  6. Mannose-rich glycosylation patterns on HIV-1 subtype C gp120 and sensitivity to the lectins, Griffithsin, Cyanovirin-N and Scytovirin

    PubMed Central

    Alexandre, Kabamba B.; Gray, Elin S.; Lambson, Bronwen E.; Moore, Penny L.; Choge, Isaac A.; Mlisana, Koleka; Abdool Karim, Salim S.; McMahon, James; O’Keefe, Barry; Chikwamba, Rachel; Morris, Lynn

    2012-01-01

    Griffithsin (GRFT), Cyanovirin-N (CV-N) and Scytovirin (SVN) are lectins that inhibit HIV-1 infection by binding to multiple mannose-rich glycans on the HIV-1 envelope glycoproteins (Env). Here we show that these lectins neutralize subtype C primary virus isolates in addition to Env-pseudotyped viruses obtained from plasma and cervical vaginal lavages. Among 15 subtype C pseudoviruses, the median IC50 values were 0.4, 1.8 and 20.1 nM for GRFT, CV-N and SVN, respectively, similar to what was found for subtype B and A. Analysis of Env sequences suggested that concomitant lack of glycans at positions 234 and 295 resulted in natural resistance to these compounds, which was confirmed by site-directed mutagenesis. Furthermore, the binding sites for these lectins overlapped that of the 2G12 monoclonal antibody epitope, which is generally absent on subtype C Env. This data support further research on these lectins as potential microbicides in the context of HIV-1 subtype C infection. PMID:20392471

  7. The lectins griffithsin, cyanovirin-N and scytovirin inhibit HIV-1 binding to the DC-SIGN receptor and transfer to CD4(+) cells.

    PubMed

    Alexandre, Kabamba B; Gray, Elin S; Mufhandu, Hazel; McMahon, James B; Chakauya, Ereck; O'Keefe, Barry R; Chikwamba, Rachel; Morris, Lynn

    2012-02-20

    It is generally believed that during the sexual transmission of HIV-1, the glycan-specific DC-SIGN receptor binds the virus and mediates its transfer to CD4(+) cells. The lectins griffithsin (GRFT), cyanovirin-N (CV-N) and scytovirin (SVN) inhibit HIV-1 infection by binding to mannose-rich glycans on gp120. We measured the ability of these lectins to inhibit both the HIV-1 binding to DC-SIGN and the DC-SIGN-mediated HIV-1 infection of CD4(+) cells. While GRFT, CV-N and SVN were moderately inhibitory to DC-SIGN binding, they potently inhibited DC-SIGN-transfer of HIV-1. The introduction of the 234 glycosylation site abolished HIV-1 sensitivity to lectin inhibition of binding to DC-SIGN and virus transfer to susceptible cells. However, the addition of the 295 glycosylation site increased the inhibition of transfer. Our data suggest that GRFT, CV-N and SVN can block two important stages of the sexual transmission of HIV-1, DC-SIGN binding and transfer, supporting their further development as microbicides. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Transformation of Althaea officinalis L. by Agrobacterium rhizogenes for the production of transgenic roots expressing the anti-HIV microbicide cyanovirin-N.

    PubMed

    Drake, Pascal M W; de Moraes Madeira, Luisa; Szeto, Tim H; Ma, Julian K-C

    2013-12-01

    The marshmallow plant (Althaea officinalis L.) has been used for centuries in medicine and other applications. Valuable secondary metabolites have previously been identified in Agrobacterium rhizogenes-generated transgenic 'hairy' roots in this species. In the present study, transgenic roots were produced in A. officinalis using A. rhizogenes. In addition to wild-type lines, roots expressing the anti-human immunodeficiency virus microbicide candidate, cyanovirin-N (CV-N), were generated. Wild-type and CV-N root lines were transferred to liquid culture and increased in mass by 49 and 19 % respectively over a 7 day culture period. In the latter, the concentration of CV-N present in the root tissue was 2.4 μg/g fresh weight, with an average secretion rate into the growth medium of 0.02 μg/ml/24 h. A. officinalis transgenic roots may therefore in the future be used not only as a source of therapeutic secondary metabolites, but also as an expression system for the production of recombinant pharmaceuticals.

  9. In vivo evaluation of safety and toxicity of a Lactobacillus jensenii producing modified cyanovirin-N in a rhesus macaque vaginal challenge model.

    PubMed

    Brichacek, Beda; Lagenaur, Laurel A; Lee, Peter P; Venzon, David; Hamer, Dean H

    2013-01-01

    Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across the cervicovaginal mucosa in women is influenced by many factors including the microbiota and the presence of underlying inflammation. It is important that potential HIV preventative agents do not alter the mucosal environment in a way that enhances HIV acquisition. We examined the impact of a "live" microbicide on the vaginal mucosal environment in a rhesus macaque repeated vaginal simian-HIV (SHIVSF162P3) challenge model. The microbicide contained a human vaginal Lactobacillus jensenii expressing the HIV-1 entry inhibitor, modified Cyanovirin-N (mCV-N), and henceforth called LB-mCV-N. Macaques were colonized vaginally each week with LB-mCV-N and sampled six days after colonization for culturable bacteria, pH and cervical-vaginal cytokines during the duration of the six-week study. We show that macaques that retained the engineered LB-mCV-N strain in their vaginal microbiota, during SHIV challenge, had lower pH, when colonization levels were higher, and had no evidence of inflammatory cytokines. Indeed, Interleukin-13, a mediator of inflammation, was detected less often in LB-mCV-N colonized macaques than in controls and we found higher levels of Interleukin 1 receptor antagonist (IL-1RA) in LB-mCV-N colonized macaques during the SHIV challenge period. We noted an inverse correlation between levels of mucosal IL-1RA and peak plasma viral load, thus higher IL-1RA correlated with lower viral load in LB-mCV-N treated macaques. These data support the use of LB-mCV-N as a safe "live" microbicide and suggest that lactobacilli themselves may positively impact the mucosal environment.

  10. In Vivo Evaluation of Safety and Toxicity of a Lactobacillus jensenii Producing Modified Cyanovirin-N in a Rhesus Macaque Vaginal Challenge Model

    PubMed Central

    Brichacek, Beda; Lagenaur, Laurel A.; Lee, Peter P.; Venzon, David; Hamer, Dean H.

    2013-01-01

    Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across the cervicovaginal mucosa in women is influenced by many factors including the microbiota and the presence of underlying inflammation. It is important that potential HIV preventative agents do not alter the mucosal environment in a way that enhances HIV acquisition. We examined the impact of a “live” microbicide on the vaginal mucosal environment in a rhesus macaque repeated vaginal simian-HIV (SHIVSF162P3) challenge model. The microbicide contained a human vaginal Lactobacillus jensenii expressing the HIV-1 entry inhibitor, modified Cyanovirin-N (mCV-N), and henceforth called LB-mCV-N. Macaques were colonized vaginally each week with LB-mCV-N and sampled six days after colonization for culturable bacteria, pH and cervical-vaginal cytokines during the duration of the six-week study. We show that macaques that retained the engineered LB-mCV-N strain in their vaginal microbiota, during SHIV challenge, had lower pH, when colonization levels were higher, and had no evidence of inflammatory cytokines. Indeed, Interleukin-13, a mediator of inflammation, was detected less often in LB-mCV-N colonized macaques than in controls and we found higher levels of Interleukin 1 receptor antagonist (IL-1RA) in LB-mCV-N colonized macaques during the SHIV challenge period. We noted an inverse correlation between levels of mucosal IL-1RA and peak plasma viral load, thus higher IL-1RA correlated with lower viral load in LB-mCV-N treated macaques. These data support the use of LB-mCV-N as a safe “live” microbicide and suggest that lactobacilli themselves may positively impact the mucosal environment. PMID:24265721

  11. Reverse genetics generation of chimeric infectious Junin/Lassa virus is dependent on interaction of homologous glycoprotein stable signal peptide and G2 cytoplasmic domains.

    PubMed

    Albariño, César G; Bird, Brian H; Chakrabarti, Ayan K; Dodd, Kimberly A; White, David M; Bergeron, Eric; Shrivastava-Ranjan, Punya; Nichol, Stuart T

    2011-01-01

    The Arenaviridae are a diverse and globally distributed collection of viruses that are maintained primarily by rodent reservoirs. Junin virus (JUNV) and Lassa virus (LASV) can both cause significant outbreaks of severe and often fatal human disease throughout their respective areas of endemicity. In an effort to improve upon the existing live attenuated JUNV Candid1 vaccine, we generated a genetically homogenous stock of this virus from cDNA copies of the virus S and L segments by using a reverse genetics system. Further, these cDNAs were used in combination with LASV cDNAs to successfully generate two recombinant Candid1 JUNV/LASV chimeric viruses (via envelope glycoprotein [GPC] exchange). It was found that while the GPC extravirion domains were readily exchangeable, homologous stable signal peptide (SSP) and G2 transmembrane and cytoplasmic tail domains were essential for correct GPC maturation and production of infectious chimeric viruses. The switching of the JUNV and LASV G1/G2 ectodomains within the Candid1 vaccine background did not alter the attenuated phenotype of the vaccine strain in a lethal mouse model. These recombinant chimeric viruses shed light on the fundamental requirements of arenavirus GPC maturation and may serve as a strategy for the development of bivalent JUNV and LASV vaccine candidates.

  12. Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver-chimeric mice from infection with a homologous hepatitis C virus strain.

    PubMed

    Vanwolleghem, Thomas; Bukh, Jens; Meuleman, Philip; Desombere, Isabelle; Meunier, Jean-Christophe; Alter, Harvey; Purcell, Robert H; Leroux-Roels, Geert

    2008-06-01

    The role of the humoral immune response in the natural course of hepatitis C virus (HCV) infection is widely debated. Most chronically infected patients have immunoglobulin G (IgG) antibodies capable of neutralizing HCV pseudoparticles (HCVpp) in vitro. It is, however, not clear whether these IgG can prevent a de novo HCV infection in vivo and contribute to the control of viremia in infected individuals. We addressed this question with homologous in vivo protection studies in human liver-urokinase-type plasminogen activator (uPA)(+/+) severe combined immune deficient (SCID) mice. Chimeric mice were loaded with chronic phase polyclonal IgG and challenged 3 days later with a 100% infectious dose of the acute phase H77C virus, both originating from patient H. Passive immunization induced sterilizing immunity in five of eight challenged animals. In the three nonprotected animals, the HCV infection was attenuated, as evidenced by altered viral kinetics in comparison with five control IgG-treated animals. Plasma samples obtained from the mice at viral challenge neutralized H77C-HCVpp at dilutions as high as 1/400. Infection was completely prevented when, before administration to naïve chimeric mice, the inoculum was pre-incubated in vitro at an IgG concentration normally observed in humans. Polyclonal IgG from a patient with a long-standing HCV infection not only displays neutralizing activity in vitro using the HCVpp system, but also conveys sterilizing immunity toward the ancestral HCV strain in vivo, using the human liver-chimeric mouse model. Both experimental systems will be useful tools to identify neutralizing antibodies for future clinical use.

  13. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    NASA Technical Reports Server (NTRS)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  14. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    NASA Technical Reports Server (NTRS)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  15. Homologous recombination and retention of a single form of most genes shape the highly chimeric mitochondrial genome of a cybrid plant.

    PubMed

    Sanchez-Puerta, M Virginia; Zubko, Mikhajlo K; Palmer, Jeffrey D

    2015-04-01

    The structure and evolution of angiosperm mitochondrial genomes are driven by extremely high rates of recombination and rearrangement. An excellent experimental system for studying these events is offered by cybrid plants, in which parental mitochondria usually fuse and their genomes recombine. Little is known about the extent, nature and consequences of mitochondrial recombination in these plants. We conducted the first study in which the organellar genomes of a cybrid - between Nicotiana tabacum and Hyoscyamus niger - were sequenced and compared to those of its parents. This cybrid mitochondrial genome is highly recombinant, reflecting at least 30 crossovers and five gene conversions between its parental genomes. It is also surprisingly large (41% and 64% larger than the parental genomes), yet contains single alleles for 90% of mitochondrial genes. Recombination produced a remarkably chimeric cybrid mitochondrial genome and occurred entirely via homologous mechanisms involving the double-strand break repair and/or break-induced replication pathways. Retention of a single form of most genes could be advantageous to minimize intracellular incompatibilities and/or reflect neutral forces that preferentially eliminate duplicated regions. We discuss the relevance of these findings to the surprisingly frequent occurrence of horizontal gene - and genome - transfer in angiosperm mitochondrial DNAs. © 2014 The Authors New Phytologist © 2014 New Phytologist Trust.

  16. NMR Solution Structure of a Cyanovirin Homolog from Wheat Head Blight Fungus

    PubMed Central

    Matei, Elena; Louis, John M.; Jee, JunGoo; Gronenborn, Angela M.

    2011-01-01

    Members of the cyanovirin-N homolog (CVNH) lectin family are found in bacteria, fungi and plants. As part of our ongoing work on CVNH structure-function studies, we determined the high-resolution NMR solution structure of the homolog from the wheat head blight disease causing ascomycetous fungus Gibberella zeae (or Fusarium graminearum), hereafter called GzCVNH. Like cyanovirin-N (CV-N), GzCVNH comprises two tandem sequence repeats and the protein sequence exhibits 30% identity with CV-N. The overall structure is similar to those of other members of the CVNH family, with the conserved pseudo-symmetric halves of the structure, domains A and B, closely resembling recently determined structures of Tuber borchii, Neurospora crassa and Ceratopteris richardii CVNH proteins. Although GzCVNH exhibits a similar glycan recognition profile to CV-N and specifically binds to Manα(1–2)Manα, its weak carbohydrate binding affinity to only one binding site is insufficient for conferring anti-HIV activity. PMID:21365681

  17. Complex chimerism

    PubMed Central

    Ma, Kimberly K.; Petroff, Margaret G.; Coscia, Lisa A.; Armenti, Vincent T.; Adams Waldorf, Kristina M.

    2013-01-01

    Thousands of women with organ transplantation have undergone successful pregnancies, however little is known about how the profound immunologic changes associated with pregnancy might influence tolerance or rejection of the allograft. Pregnant women with a solid organ transplant are complex chimeras with multiple foreign cell populations from the donor organ, fetus, and mother of the pregnant woman. We consider the impact of complex chimerism and pregnancy-associated immunologic changes on tolerance of the allograft both during pregnancy and the postpartum period. Mechanisms of allograft tolerance are likely dynamic during pregnancy and affected by the influx of fetal microchimeric cells, HLA relationships (between the fetus, pregnant woman and/or donor), peripheral T cell tolerance to fetal cells, and fetal minor histocompatibility antigens. Further research is necessary to understand the complex immunology during pregnancy and the postpartum period of women with a solid organ transplant. PMID:23974274

  18. Chimeric Pestivirus Experimental Vaccines.

    PubMed

    Reimann, Ilona; Blome, Sandra; Beer, Martin

    2016-01-01

    Chimeric pestiviruses have shown great potential as marker vaccine candidates against pestiviral infections. Exemplarily, we describe here the construction and testing of the most promising classical swine fever vaccine candidate "CP7_E2alf" in detail. The description is focused on classical cloning technologies in combination with reverse genetics.

  19. Engineering Chimeric Antigen Receptors

    PubMed Central

    Kulemzin, S. V.; Kuznetsova, V. V.; Mamonkin, M.; Taranin, A. V.; Gorchakov, A. A.

    2017-01-01

    Chimeric antigen receptors (CARs) are recombinant protein molecules that redirect cytotoxic lymphocytes toward malignant and other target cells. The high feasibility of manufacturing CAR-modified lymphocytes for the therapy of cancer has spurred the development and optimization of new CAR T cells directed against a broad range of target antigens. In this review, we describe the main structural and functional elements constituting a CAR, discuss the roles of these elements in modulating the anti-tumor activity of CAR T cells, and highlight alternative approaches to CAR engineering. PMID:28461969

  20. Development of a chimeric DNA-RNA hammerhead ribozyme targeting SARS virus.

    PubMed

    Fukushima, Akiko; Fukuda, Noboru; Lai, Yimu; Ueno, Takahiro; Moriyama, Mitsuhiko; Taguchi, Fumihiro; Iguchi, Akifumi; Shimizu, Kazushi; Kuroda, Kazumichi

    2009-01-01

    Severe acute respiratory syndrome (SARS) is a severe pulmonary infectious disease caused by a novel coronavirus. To develop an effective and specific medicine targeting the SARS-coronavirus (CoV), a chimeric DNA-RNA hammerhead ribozyme was designed and synthesized using a sequence homologous with the mouse hepatitis virus (MHV). Chimeric DNA-RNA hammerhead ribozyme targeting MHV and SARS-CoV were designed and synthesized.To confirm its activity, in vitro cleavage reactions were performed with the synthesized ribozyme. Effects of the chimeric ribozyme were evaluated on multiplication of MHV. Effects of the chimeric ribozyme on expression of SARS-CoV were evaluated in cultured 3T3 cells. The synthetic ribozyme cleaved the synthetic target MHV and SARS-CoV RNA into fragments of predicted length. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited multiplication of MHV in DBT cells by about 60%. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited the expression of SARS-CoV RNA in 3T3 cells transfected with the recombinant plasmid. The chimeric DNA-RNA ribozyme targeting SARS-CoV significantly inhibited MHV viral activity and expression of recombinant SARS RNA in vitro. These findings indicate that the synthetic chimeric DNA-RNA ribozyme could provide a feasible treatment for SARS. Copyright (c) 2009 S. Karger AG, Basel.

  1. Alcohol homologation

    DOEpatents

    Wegman, R.W.; Moloy, K.G.

    1988-02-23

    A process is described for the homologation of an alkanol by reaction with synthesis gas in contact with a system containing rhodium atom, ruthenium atom, iodine atom and a bis(diorganophosphino) alkane to selectivity produce the next higher homologue.

  2. Alcohol homologation

    DOEpatents

    Wegman, Richard W.; Moloy, Kenneth G.

    1988-01-01

    A process for the homologation of an alkanol by reaction with synthesis gas in contact with a system containing rhodium atom, ruthenium atom, iodine atom and a bis(diorganophosphino) alkane to selectivity produce the next higher homologue.

  3. Invasiveness, chimerism and genetic diversity.

    PubMed

    Ben-Shlomo, Rachel

    2017-09-26

    Adaptation for invasiveness should comprise the capability to exploit and prosper in a wide range of ecological conditions, and is therefore expected to be associated with a certain level of genetic diversity. Paradoxically, however, invasive populations are established by only a few founders, resulting in low genetic diversity. As a conceivable way of attaining high genetic diversity and high variance of gene expression even when a small number of founders is involved in invasiveness, I suggest here chimerism, a fusion between different individuals-a common phenomenon found in numerous phyla. The composite entity offers the chimeric organism genetic flexibility and higher inclusive fitness that depends on the joint genomic fitness of the original partners. The ability to form a chimeric entity is also applied to subsequent generations and, consequently, the level of genetic diversity does not decline over generations of population establishment following invasion. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  4. How should chimerism be decoded?

    PubMed

    Ferrand, Christophe; Perruche, Sylvain; Robinet, Eric; Martens, Anton; Tiberghien, Pierre; Saas, Philippe

    2003-05-15

    To date, the significance of chimerism has not been fully understood. In particular, microchimerism can be associated with allograft acceptance or rejection. Several factors may influence the immunologic consequences of chimerism. In this review, the major factors influencing these consequences are briefly described. Subsequently, the different methods available for detecting and tracking donor-derived cells are listed. These techniques have been mainly developed concomitantly with nonmyeloablative hematopoietic allografts to monitor immunosuppression. Finally, the authors suggest how these methods may help to improve the understanding of microchimerism in solid organ transplantation.

  5. Chimeric enzymes with improved cellulase activities

    DOEpatents

    Xu, Qi; Baker, John O; Himmel, Michael E

    2015-03-31

    Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

  6. Targeting duplex DNA with chimeric α,β-triplex-forming oligonucleotides

    PubMed Central

    Kolganova, N. A.; Shchyolkina, A. K.; Chudinov, A. V.; Zasedatelev, A. S.; Florentiev, V. L.; Timofeev, E. N.

    2012-01-01

    Triplex-directed DNA recognition is strictly limited by polypurine sequences. In an attempt to address this problem with synthetic biology tools, we designed a panel of short chimeric α,β-triplex-forming oligonucleotides (TFOs) and studied their interaction with fluorescently labelled duplex hairpins using various techniques. The hybridization of hairpin with an array of chimeric probes suggests that recognition of double-stranded DNA follows complicated rules combining reversed Hoogsteen and non-canonical homologous hydrogen bonding. In the presence of magnesium ions, chimeric TFOs are able to form highly stable α,β-triplexes, as indicated by native gel-electrophoresis, on-array thermal denaturation and fluorescence-quenching experiments. CD spectra of chimeric triplexes exhibited features typically observed for anti-parallel purine triplexes with a GA or GT third strand. The high potential of chimeric α,β-TFOs in targeting double-stranded DNA was demonstrated in the EcoRI endonuclease protection assay. In this paper, we report, for the first time, the recognition of base pair inversions in a duplex by chimeric TFOs containing α-thymidine and α-deoxyguanosine. PMID:22641847

  7. Mechanisms of tolerance induced via mixed chimerism.

    PubMed

    Sykes, Megan

    2007-05-01

    Mixed hematopoietic chimerism provides a powerful means of inducing robust, donor-specific tolerance. In this article, the minimal requirements for achieving mixed chimerism, the development of new reagents that promote its achievement, and the mechanisms by which peripheral and intrathymic tolerance are achieved via mixed chimerism are discussed. An emerging understanding of these mechanisms, along with the development of new immunosuppressive reagents, is allowing advancement toward clinical application of this approach.

  8. Theoretical design of a new chimeric protein for the treatment of breast cancer.

    PubMed

    Soleimani, Meysam; Mahnam, Karim; Mirmohammad-Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Jahanian-Najafabadi, Ali

    2016-01-01

    p28 and NRC peptides are two anticancer peptides with various mechanisms have shown to be effective against breast cancer. Therefore, it seems that construction of a chimeric protein containing the two peptides might cause synergistic cytotoxic effects. However, since the two peptides bear opposite charges, production of a chimeric protein in which the two moieties do not intervene each other is difficult. In this study, our goal was to find a suitable peptide linker for the new chimeric protein in a manner that none of the peptides intervene the other's function. We selected some linkers with different characteristics and lengths and created a small library of the chimeric proteins harboring these linkers. Homology modeling and molecular dynamic simulation revealed that (PA)5P and (EAAAK)3 linkers can separate the p28 and NRC peptides effectively. Thus, the chimeric protein linked with (PA)5P or (EAAAK)3 linkers might show synergistic and stronger anticancer effects than the separate peptide moieties because they could exert their cytotoxic effects freely which is not influenced by the other part.

  9. Theoretical design of a new chimeric protein for the treatment of breast cancer

    PubMed Central

    Soleimani, Meysam; Mahnam, Karim; Mirmohammad-Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Jahanian-Najafabadi, Ali

    2016-01-01

    p28 and NRC peptides are two anticancer peptides with various mechanisms have shown to be effective against breast cancer. Therefore, it seems that construction of a chimeric protein containing the two peptides might cause synergistic cytotoxic effects. However, since the two peptides bear opposite charges, production of a chimeric protein in which the two moieties do not intervene each other is difficult. In this study, our goal was to find a suitable peptide linker for the new chimeric protein in a manner that none of the peptides intervene the other’s function. We selected some linkers with different characteristics and lengths and created a small library of the chimeric proteins harboring these linkers. Homology modeling and molecular dynamic simulation revealed that (PA)5P and (EAAAK)3 linkers can separate the p28 and NRC peptides effectively. Thus, the chimeric protein linked with (PA)5P or (EAAAK)3 linkers might show synergistic and stronger anticancer effects than the separate peptide moieties because they could exert their cytotoxic effects freely which is not influenced by the other part. PMID:27499788

  10. Chimeric Foot-and-Mouth Disease Viruses: Evaluation of Their Efficacy as Potential Marker Vaccines in Cattle

    USDA-ARS?s Scientific Manuscript database

    Previous work in swine has demonstrated that full protection against Foot-and-Mouth Disease (FMD) can be achieved following vaccination with chimeric Foot-and-Mouth Disease Virus (FMDV) vaccines, whereby the VP1 G-H loop has been substituted with a non-homologous alternative. If proven to be effect...

  11. Homology and causes.

    PubMed

    Van Valen, L M

    1982-09-01

    Homology is resemblance caused by a continuity of information. In biology it is a unified developmental phenomenon. Homologies among and within individuals intergrade in several ways, so historical homology cannot be separated sharply from repetitive homology. Nevertheless, the consequences of historical and repetitive homologies can be mutually contradictory. A detailed discussion of the rise and fall of the "premolar-analogy" theory of homologies of mammalian molar-tooth cusps exemplifies such a contradiction. All other hypotheses of historical homology which are based on repetitive homology, such as the foliar theory of the flower considered phyletically, are suspect.

  12. Custom-engineered chimeric foot-and-mouth disease vaccine elicits protective immune responses in pigs.

    PubMed

    Blignaut, Belinda; Visser, Nico; Theron, Jacques; Rieder, Elizabeth; Maree, Francois F

    2011-04-01

    Chimeric foot-and-mouth disease viruses (FMDV) of which the antigenic properties can be readily manipulated is a potentially powerful approach in the control of foot-and-mouth disease (FMD) in sub-Saharan Africa. FMD vaccine application is complicated by the extensive variability of the South African Territories (SAT) type viruses, which exist as distinct genetic and antigenic variants in different geographical regions. A cross-serotype chimeric virus, vKNP/SAT2, was engineered by replacing the external capsid-encoding region (1B-1D/2A) of an infectious cDNA clone of the SAT2 vaccine strain, ZIM/7/83, with that of SAT1 virus KNP/196/91. The vKNP/SAT2 virus exhibited comparable infection kinetics, virion stability and antigenic profiles to the KNP/196/91 parental virus, thus indicating that the functions provided by the capsid can be readily exchanged between serotypes. As these qualities are necessary for vaccine manufacturing, high titres of stable chimeric virus were obtained. Chemically inactivated vaccines, formulated as double-oil-in-water emulsions, were produced from intact 146S virion particles of both the chimeric and parental viruses. Inoculation of guinea pigs with the respective vaccines induced similar antibody responses. In order to show compliance with commercial vaccine requirements, the vaccines were evaluated in a full potency test. Pigs vaccinated with the chimeric vaccine produced neutralizing antibodies and showed protection against homologous FMDV challenge, albeit not to the same extent as for the vaccine prepared from the parental virus. These results provide support that chimeric vaccines containing the external capsid of field isolates can be successfully produced and that they induce protective immune responses in FMD host species.

  13. Homology, Analogy, and Ethology.

    ERIC Educational Resources Information Center

    Beer, Colin G.

    1984-01-01

    Because the main criterion of structural homology (the principle of connections) does not exist for behavioral homology, the utility of the ethological concept of homology has been questioned. The confidence with which behavioral homologies can be claimed varies inversely with taxonomic distance. Thus, conjectures about long-range phylogenetic…

  14. Homology, Analogy, and Ethology.

    ERIC Educational Resources Information Center

    Beer, Colin G.

    1984-01-01

    Because the main criterion of structural homology (the principle of connections) does not exist for behavioral homology, the utility of the ethological concept of homology has been questioned. The confidence with which behavioral homologies can be claimed varies inversely with taxonomic distance. Thus, conjectures about long-range phylogenetic…

  15. Exploration of genetically encoded voltage indicators based on a chimeric voltage sensing domain.

    PubMed

    Mishina, Yukiko; Mutoh, Hiroki; Song, Chenchen; Knöpfel, Thomas

    2014-01-01

    Deciphering how the brain generates cognitive function from patterns of electrical signals is one of the ultimate challenges in neuroscience. To this end, it would be highly desirable to monitor the activities of very large numbers of neurons while an animal engages in complex behaviors. Optical imaging of electrical activity using genetically encoded voltage indicators (GEVIs) has the potential to meet this challenge. Currently prevalent GEVIs are based on the voltage-sensitive fluorescent protein (VSFP) prototypical design or on the voltage-dependent state transitions of microbial opsins. We recently introduced a new VSFP design in which the voltage-sensing domain (VSD) is sandwiched between a fluorescence resonance energy transfer pair of fluorescent proteins (termed VSFP-Butterflies) and also demonstrated a series of chimeric VSD in which portions of the VSD of Ciona intestinalis voltage-sensitive phosphatase are substituted by homologous portions of a voltage-gated potassium channel subunit. These chimeric VSD had faster sensing kinetics than that of the native Ci-VSD. Here, we describe a new set of VSFPs that combine chimeric VSD with the Butterfly structure. We show that these chimeric VSFP-Butterflies can report membrane voltage oscillations of up to 200 Hz in cultured cells and report sensory evoked cortical population responses in living mice. This class of GEVIs may be suitable for imaging of brain rhythms in behaving mammalians.

  16. Chimeric pneumococcal cell wall lytic enzymes reveal important physiological and evolutionary traits.

    PubMed

    Diaz, E; López, R; Garcia, J L

    1991-03-25

    Two novel chimeric pneumococcal cell wall lytic enzymes, named LC7 and CL7, have been constructed by in vitro recombination of the lytA gene encoding the major autolysin (LYTA amidase) of Streptococcus pneumoniae, a choline-dependent enzyme, and the cpl7 gene encoding the CPL7 lysozyme of phage Cp-7, a choline-independent enzyme. In remarkable contrast with previous chimeric constructions, we fused here two genes that lack nucleotide homology. The CL7 enzyme, which contains the N-terminal domain of CPL7 and C-terminal domain of LYTA, exhibited a choline-dependent lysozyme activity. This experimental rearrangement of domains might mimic the process that have generated the choline-dependent CPL1 lysozyme of phage Cp-1 during evolution, providing additional support to the modular theory of protein evolution. The LC7 enzyme, built up by fusion of the N-terminal domain of LYTA and the C-terminal domain of CPL7, exhibited an amidase activity capable of degrading ethanolamine-containing cell walls. The chimeric amidase behaved as an autolytic enzyme when it was cloned and expressed in S. pneumoniae. The chimeric enzymes provided new insights on the mechanisms involved in regulation of the host pneumococcal autolysins and on the participation of these enzymes in the process of cell separation. Furthermore, our experimental approach confirmed the basic role of the C-terminal domains in substrate recognition and revealed the influence of these domains on the optimal pH for catalytic activity.

  17. Intra-serotype SAT2 chimeric foot-and-mouth disease vaccine protects cattle against FMDV challenge.

    PubMed

    Maree, Francois F; Nsamba, Peninah; Mutowembwa, Paidamwoyo; Rotherham, Lia S; Esterhuysen, Jan; Scott, Katherine

    2015-06-09

    The genetic diversity of the three Southern African Territories (SAT) types of foot-and-mouth disease virus (FMDV) reflects high antigenic variation, and indications are that vaccines targeting each SAT-specific topotype may be needed. This has serious implications for control of FMD using vaccines as well as the choice of strains to include in regional antigen banks. Here, we investigated an intra-serotype chimeric virus, vSAT2(ZIM14)-SAT2, which was engineered by replacing the surface-exposed capsid-coding region (1B-1D/2A) of a SAT2 genome-length clone, pSAT2, with that of the field isolate, SAT2/ZIM/14/90. The chimeric FMDV produced by this technique was viable, grew to high titres and stably maintained the 1B-1D/2A sequence upon passage. Chemically inactivated, oil adjuvanted vaccines of both the chimeric and parental immunogens were used to vaccinate cattle. The serological response to vaccination showed the production of strong neutralizing antibody titres that correlated with protection against homologous FMDV challenge. We also predicted a good likelihood that cattle vaccinated with an intra-serotype chimeric vaccine would be protected against challenge with viruses that caused recent outbreaks in southern Africa. These results provide support that chimeric vaccines containing the external capsid of field isolates induce protective immune responses in FMD host species similar to the parental vaccine.

  18. Chimeric polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Wogulis, Mark; Sweeney, Matthew; Heu, Tia

    2017-06-14

    The present invention relates to chimeric GH61 polypeptides having cellulolytic enhancing activity. The present invention also relates to polynucleotides encoding the chimeric GH61 polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the chimeric GH61 polypeptides.

  19. Nitrogenase and Homologs

    PubMed Central

    2014-01-01

    Nitrogenase catalyzes biological nitrogen fixation, a key step in the global nitrogen cycle. Three homologous nitrogenases have been identified to date, along with several structural and/or functional homologs of this enzyme that are involved in nitrogenase assembly, bacteriochlorophyll biosynthesis and methanogenic process, respectively. In this article, we provide an overview of the structures and functions of nitrogenase and its homologs, which highlights the similarity and disparity of this uniquely versatile group of enzymes. PMID:25491285

  20. Quick selection of a chimeric T2 phage that displays active enzyme on the viral capsid.

    PubMed

    Tanji, Yasunori; Murofushi, Keita; Miyanaga, Kazuhiko

    2005-01-01

    We designed a bacteriophage T2 system to display proteins fused at the N-terminus of the head protein small outer capsid (SOC) of a T2 phage. To facilitate selection of chimeric phage, a T2 phage encoding the beta-galactosidase gene (betagal) upstream of the soc gene was constructed. The phage, named T2betaGal, produces blue plaques on agar plates containing XGal. Subsequently, a plasmid encoding the target protein upstream of soc was constructed and used to transform E. coli B(E) cells. Transformed cells were infected with T2betaGal and homologous recombination between phage DNA and the plasmid resulted in a chimeric phage that produced transparent plaques due to the excision of the betagal gene. Chitosanase of Bacillus sp. strain K17 (ChoK), consisting of 453 amino acids, was used as a model target protein. Recombinant T2 phage that produced ChoK was named T2ChoK. T2ChoK was produced from T2betaGal at a recombination frequency of about 0.1%. On the other hand, the value for T2betaGal produced from wild-type T2 was 0.001 %. This new system enables us to select recombinant phage very quickly and accurately. The number of molecules of ChoK was calculated at 14.7 per single phage. Latent period and burst size were estimated for the chimeric phages.

  1. Homological stabilizer codes

    SciTech Connect

    Anderson, Jonas T.

    2013-03-15

    In this paper we define homological stabilizer codes on qubits which encompass codes such as Kitaev's toric code and the topological color codes. These codes are defined solely by the graphs they reside on. This feature allows us to use properties of topological graph theory to determine the graphs which are suitable as homological stabilizer codes. We then show that all toric codes are equivalent to homological stabilizer codes on 4-valent graphs. We show that the topological color codes and toric codes correspond to two distinct classes of graphs. We define the notion of label set equivalencies and show that under a small set of constraints the only homological stabilizer codes without local logical operators are equivalent to Kitaev's toric code or to the topological color codes. - Highlights: Black-Right-Pointing-Pointer We show that Kitaev's toric codes are equivalent to homological stabilizer codes on 4-valent graphs. Black-Right-Pointing-Pointer We show that toric codes and color codes correspond to homological stabilizer codes on distinct graphs. Black-Right-Pointing-Pointer We find and classify all 2D homological stabilizer codes. Black-Right-Pointing-Pointer We find optimal codes among the homological stabilizer codes.

  2. Transcriptome Sequencing for the Detection of Chimeric Transcripts.

    PubMed

    Chu, Hsueh-Ting

    2016-01-01

    The occurrence of chimeric transcripts has been reported in many cancer cells and seen as potential biomarkers and therapeutic targets. Modern high-throughput sequencing technologies offer a way to investigate individual chimeric transcripts and the systematic information of associated gene expressions about underlying genome structural variations and genomic interactions. The detection methods of finding chimeric transcripts from massive amount of short read sequence data are discussed here. Both assembly-based and alignment-based methods are used for the investigation of chimeric transcripts.

  3. Humanization of excretory pathway in chimeric mice with humanized liver.

    PubMed

    Okumura, Hirotoshi; Katoh, Miki; Sawada, Toshiro; Nakajima, Miki; Soeno, Yoshinori; Yabuuchi, Hikaru; Ikeda, Toshihiko; Tateno, Chise; Yoshizato, Katsutoshi; Yokoi, Tsuyoshi

    2007-06-01

    The liver of a chimeric urokinase-type plasminogen activator (uPA)(+/+)/severe combined immunodeficient (SCID) mouse line recently established in Japan could be replaced by more than 80% with human hepatocytes. We previously reported that the chimeric mice with humanized liver could be useful as a human model in studies on drug metabolism and pharmacokinetics. In the present study, the humanization of an excretory pathway was investigated in the chimeric mice. Cefmetazole (CMZ) was used as a probe drug. The CMZ excretions in urine and feces were 81.0 and 5.9% of the dose, respectively, in chimeric mice and were 23.7 and 59.4% of the dose, respectively, in control uPA(-/-)/SCID mice. Because CMZ is mainly excreted in urine in humans, the excretory profile of chimeric mice was demonstrated to be similar to that of humans. In the chimeric mice, the hepatic mRNA expression of human drug transporters could be quantified. On the other hand, the hepatic mRNA expression of mouse drug transporters in the chimeric mice was significantly lower than in the control uPA(-/-)/SCID mice. In conclusion, chimeric mice exhibited a humanized profile of drug excretion, suggesting that this chimeric mouse line would be a useful animal model in excretory studies.

  4. Homology, convergence and parallelism.

    PubMed

    Ghiselin, Michael T

    2016-01-05

    Homology is a relation of correspondence between parts of parts of larger wholes. It is used when tracking objects of interest through space and time and in the context of explanatory historical narratives. Homologues can be traced through a genealogical nexus back to a common ancestral precursor. Homology being a transitive relation, homologues remain homologous however much they may come to differ. Analogy is a relationship of correspondence between parts of members of classes having no relationship of common ancestry. Although homology is often treated as an alternative to convergence, the latter is not a kind of correspondence: rather, it is one of a class of processes that also includes divergence and parallelism. These often give rise to misleading appearances (homoplasies). Parallelism can be particularly hard to detect, especially when not accompanied by divergences in some parts of the body. © 2015 The Author(s).

  5. Homology, convergence and parallelism

    PubMed Central

    Ghiselin, Michael T.

    2016-01-01

    Homology is a relation of correspondence between parts of parts of larger wholes. It is used when tracking objects of interest through space and time and in the context of explanatory historical narratives. Homologues can be traced through a genealogical nexus back to a common ancestral precursor. Homology being a transitive relation, homologues remain homologous however much they may come to differ. Analogy is a relationship of correspondence between parts of members of classes having no relationship of common ancestry. Although homology is often treated as an alternative to convergence, the latter is not a kind of correspondence: rather, it is one of a class of processes that also includes divergence and parallelism. These often give rise to misleading appearances (homoplasies). Parallelism can be particularly hard to detect, especially when not accompanied by divergences in some parts of the body. PMID:26598721

  6. Immunizations with chimeric hepatitis B virus-like particles to induce potential anti-hepatitis C virus neutralizing antibodies.

    PubMed

    Vietheer, Patricia T K; Boo, Irene; Drummer, Heidi E; Netter, Hans-Jürgen

    2007-01-01

    Virus-like particles (VLPs) are highly immunogenic and proven to induce protective immunity. The small surface antigen (HBsAg-S) of hepatitis B virus (HBV) self-assembles into VLPs and its use as a vaccine results in protective antiviral immunity against HBV infections. Chimeric HBsAg-S proteins carrying foreign epitopes allow particle formation and have the ability to induce anti-foreign humoral and cellular immune responses. The insertion of the hypervariable region 1 (HVR1) sequence derived from the envelope protein 2 (E2) of hepatitis C virus (HCV) into the major antigenic site of HBsAg-S ('a'-determinant) resulted in the formation of highly immunogenic VLPs that retained the antigenicity of the inserted HVR1 sequence. BALB/c mice were immunized with chimeric VLPs, which resulted in antisera with anti-HCV activity. The antisera were able to immunoprecipitate native HCV envelope complexes (E1E2) containing homologous or heterologous HVR1 sequences. HCV E1E2 pseudotyped HIV-1 particles (HCVpp) were used to measure entry into HuH-7 target cells in the presence or absence of antisera that were raised against chimeric VLPs. Anti-HVR1 VLP sera interfered with entry of entry-competent HCVpps containing either homologous or heterologous HVR1 sequences. Also, immunizations with chimeric VLPs induced antisurface antigen (HBsAg) antibodies, indicating that HBV-specific antigenicity and immunogenicity of the 'a'-determinant region is retained. A multivalent vaccine against different pathogens based on the HBsAg delivery platform should be possible. We hypothesize that custom design of VLPs with an appropriate set of HCV-neutralizing epitopes will induce antibodies that would serve to decrease the viral load at the initial infecting inoculum.

  7. Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

    PubMed

    Wu, Jun; Platero-Luengo, Aida; Sakurai, Masahiro; Sugawara, Atsushi; Gil, Maria Antonia; Yamauchi, Takayoshi; Suzuki, Keiichiro; Bogliotti, Yanina Soledad; Cuello, Cristina; Morales Valencia, Mariana; Okumura, Daiji; Luo, Jingping; Vilariño, Marcela; Parrilla, Inmaculada; Soto, Delia Alba; Martinez, Cristina A; Hishida, Tomoaki; Sánchez-Bautista, Sonia; Martinez-Martinez, M Llanos; Wang, Huili; Nohalez, Alicia; Aizawa, Emi; Martinez-Redondo, Paloma; Ocampo, Alejandro; Reddy, Pradeep; Roca, Jordi; Maga, Elizabeth A; Esteban, Concepcion Rodriguez; Berggren, W Travis; Nuñez Delicado, Estrella; Lajara, Jeronimo; Guillen, Isabel; Guillen, Pedro; Campistol, Josep M; Martinez, Emilio A; Ross, Pablo Juan; Izpisua Belmonte, Juan Carlos

    2017-01-26

    Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

  8. Lung rejection occurs in lung transplant recipients with blood chimerism.

    PubMed

    Knoop, C; Andrien, M; Defleur, V; Antoine, M; de Francquen, P; Goldman, M; Estenne, M

    1997-07-15

    It has been postulated that chimerism after transplantation might promote graft acceptance. In the present study, we prospectively assessed blood chimerism in 10 lung transplant recipients during the first posttransplant year and investigated whether chimerism was associated with an immunologically stable situation of the graft. The recipients' peripheral blood mononuclear cells were obtained before transplantation and at various time points during the first postoperative year. Donor cells were detected using nested polymerase chain reaction amplification of a donor-specific HLA-DRB1 allele. Clinical graft acceptance was determined by the number of rejection episodes. The incidence of blood chimerism was high during the first 3 postoperative months and then decreased over time. All patients experienced at least one acute rejection episode, and three patients developed chronic rejection. We, thus, conclude that rejection of the lung allograft may occur in the presence of blood chimerism.

  9. Braid Floer homology

    NASA Astrophysics Data System (ADS)

    van den Berg, J. B.; Ghrist, R.; Vandervorst, R. C.; Wójcik, W.

    2015-09-01

    Area-preserving diffeomorphisms of a 2-disc can be regarded as time-1 maps of (non-autonomous) Hamiltonian flows on R / Z ×D2. The periodic flow-lines define braid (conjugacy) classes, up to full twists. We examine the dynamics relative to such braid classes and define a new invariant for such classes, the BRAID FLOER HOMOLOGY. This refinement of Floer homology, originally used for the Arnol'd Conjecture, yields a Morse-type forcing theory for periodic points of area-preserving diffeomorphisms of the 2-disc based on braiding. Contributions of this paper include (1) a monotonicity lemma for the behavior of the nonlinear Cauchy-Riemann equations with respect to algebraic lengths of braids; (2) establishment of the topological invariance of the resulting braid Floer homology; (3) a shift theorem describing the effect of twisting braids in terms of shifting the braid Floer homology; (4) computation of examples; and (5) a forcing theorem for the dynamics of Hamiltonian disc maps based on braid Floer homology.

  10. DNA shuffling of uricase gene leads to a more "human like" chimeric uricase with increased uricolytic activity.

    PubMed

    Chen, Jing; Jiang, Nan; Wang, Tao; Xie, Guangrong; Zhang, Zhilai; Li, Hui; Yuan, Jing; Sun, Zengxian; Chen, Jianhua

    2016-01-01

    Urate oxidase (Uox) is the enzyme involved in purine metabolism. Pseudogenization of Uox gene is the underlying mechanism of hyperuricemia and gout in human. Although Uox from various microorganisms has been used in clinical practice for many years, its application is limited by potential immunogenicity. In order to develop a more "human like" uricase, DNA shuffling was used to create chimeric uricase with both improved enzymatic activity and increased homology with deduced human uricase (dHU) gene. By using wild porcine uricase (wPU) gene and dhu as parental genes, a diverse chimeric library was generated. After preliminary screening by a "homebrew" high throughput protocol, approximately 100 chimeras with relatively high enzymatic activity were obtained. By further activity comparison of the purified enzymes, chimera-62 with increase in both activity and homology with dHU compared with wPU was selected. Its Km and catalytic efficiency were determined as 9.43±0.04μM and 2.67s(-1)μM(-1) respectively. There were 33 amino acid substitutions in chimera-62 when compared with dHU and 5 substitutions when compared with wPU. By homology modeling and 3-D structure analysis, it was speculated that mutations G248S and L266F contributed to the increased activity of chimera-62 by increasing the stability of α-helix and surface polarity respectively.

  11. Vectors expressing chimeric Japanese encephalitis dengue 2 viruses.

    PubMed

    Wei, Y; Wang, S; Wang, X

    2014-01-01

    Vectors based on self-replicating RNAs (replicons) of flaviviruses are becoming powerful tool for expression of heterologous genes in mammalian cells and development of novel antiviral and anticancer vaccines. We constructed two vectors expressing chimeric viruses consisting of attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) in which the PrM/M-E genes were replaced fully or partially with those of dengue 2 virus (DENV-2). These vectors, named pJED2 and pJED2-1770 were transfected to BHK-21 cells and produced chimeric viruses JED2V and JED2-1770V, respectively. The chimeric viruses could be passaged in C6/36 but not BHK-21 cells. The chimeric viruses produced in C6/36 cells CPE 4-5 days after infection and RT-PCR, sequencing, immunofluorescence assay (IFA) and Western blot analysis confirmed the chimeric nature of produced viruses. The immunogenicity of chimeric viruses in mice was proved by detecting DENV-2 E protein-specific serum IgG antibodies with neutralization titer of 10. Successful preparation of infectious clones of chimeric JEV-DENV-2 viruses showed that JEV-based expression vectors are fully functional.

  12. chimeraviz: a tool for visualizing chimeric RNA.

    PubMed

    Lågstad, Stian; Zhao, Sen; Hoff, Andreas M; Johannessen, Bjarne; Lingjærde, Ole Christian; Skotheim, Rolf I

    2017-09-15

    Advances in high-throughput RNA sequencing have enabled more efficient detection of fusion transcripts, but the technology and associated software used for fusion detection from sequencing data often yield a high false discovery rate. Good prioritization of the results is important, and this can be helped by a visualization framework that automatically integrates RNA data with known genomic features. Here we present chimeraviz , a Bioconductor package that automates the creation of chimeric RNA visualizations. The package supports input from nine different fusion-finder tools: deFuse, EricScript, InFusion, JAFFA, FusionCatcher, FusionMap, PRADA, SOAPfuse and STAR-FUSION. chimeraviz is an R package available via Bioconductor ( https://bioconductor.org/packages/release/bioc/html/chimeraviz.html ) under Artistic-2.0. Source code and support is available at GitHub ( https://github.com/stianlagstad/chimeraviz ). rolf.i.skotheim@rr-research.no. Supplementary data are available at Bioinformatics online.

  13. Homology recognition funnel

    NASA Astrophysics Data System (ADS)

    Lee, Dominic; Kornyshev, Alexei A.

    2009-10-01

    The recognition of homologous sequences of DNA before strand exchange is considered to be the most puzzling stage of homologous recombination. A mechanism for two homologous dsDNAs to recognize each other from a distance in electrolytic solution without unzipping had been proposed in an earlier paper [A. A. Kornyshev and S. Leikin, Phys. Rev. Lett. 86, 366 (2001)]. In that work, the difference in the electrostatic interaction energy between homologous duplexes and between nonhomologous duplexes, termed the recognition energy, has been calculated. That calculation was later extended in a series of papers to account for torsional elasticity of the molecules. A recent paper [A. A. Kornyshev and A. Wynveen, Proc. Natl. Acad. Sci. U.S.A. 106, 4683 (2009)] investigated the form of the potential well that homologous DNA molecules may feel when sliding along each other. A simple formula for the shape of the well was obtained. However, this latter study was performed under the approximation that the sliding molecules are torsionally rigid. Following on from this work, in the present article we investigate the effect of torsional flexibility of the molecules on the shape of the well. A variational approach to this problem results in a transcendental equation that is easily solved numerically. Its solutions show that at large interaxial separations the recognition well becomes wider and shallower, whereas at closer distances further unexpected features arise related to an abrupt change in the mean azimuthal alignment of the molecules. The energy surface as a function of interaxial separation and the axial shift defines what we call the recognition funnel. We show that it depends dramatically on the patterns of adsorption of counterions on DNA.

  14. Structure-Activity Relationship and Signaling of New Chimeric CXCR4 Agonists.

    PubMed

    Mona, Christine E; Besserer-Offroy, Élie; Cabana, Jérôme; Lefrançois, Marilou; Boulais, Philip E; Lefebvre, Marie-Reine; Leduc, Richard; Lavigne, Pierre; Heveker, Nikolaus; Marsault, Éric; Escher, Emanuel

    2016-08-25

    The CXCR4 receptor binds with meaningful affinities only CXCL12 and synthetic antagonists/inverse agonists. We recently described high affinity synthetic agonists for this chemokine receptor, obtained by grafting the CXCL12 N-terminus onto the inverse agonist T140. While those chimeric molecules behave as agonists for CXCR4, their binding and activation mode are unknown. The present SAR of those CXCL12-oligopeptide grafts reveals the key determinants involved in CXCR4 activation. Position 3 (Val) controls affinity, whereas position 7 (Tyr) acts as an efficacy switch. Chimeric molecules bearing aromatic residues in position 3 possess high binding affinities for CXCR4 and are Gαi full agonists with robust chemotactic properties. Fine-tuning of electron-poor aromatic rings in position 7 enhances receptor activation. To rationalize these results, a homology model of a receptor-ligand complex was built using the published crystal structures of CXCR4. Molecular dynamics simulations reveal further details accounting for the observed SAR for this series.

  15. Assembly and immunogenicity of chimeric Gag-Env proteins derived from the human immunodeficiency virus type 1.

    PubMed

    Truong, C; Brand, D; Mallet, F; Roingeard, P; Brunet, S; Barin, F

    1996-03-01

    We evaluated the potential of the precursor Gag protein (Pr55) of the human immunodeficiency virus type 1 (HIV-1) as a carrier for the presentation of envelope epitopes. Recombinant chimeric core-envelope protein-expressing constructs were derived by deletion of regions within the gag gene, especially of regions encoding p24 capsid epitopes. Sequences encoding either the principal neutralization determinant (PND) and/or the CD4-binding domains (CD4BS) were then inserted. Deletion of residues 196-226 within the p24 capsid protein did not prevent self-assembly into virus-like particles (VLPs) whereas deletion of residues 299-328 completely abolished VLP formation. Thus the major homology region (MHR) and proximal sequences are required for capsid assembly. An immunization study in mice showed that assembled chimeric proteins elicited strong anti-Gag, weak anti-envelope, and no neutralizing humoral responses. Nonassembled chimeric proteins were poor immunogens. Mapping of Pr55 antigenic sites using sera from immunized mice and peptides overlapping the entire Gag precursor showed that p24 capsid and p17 matrix epitopes presented to the immune system differed from the mature form (p24 or p17) and the multimeric immature form (Pr55).

  16. Functional participation of a nifH-arsA2 chimeric fusion gene in arsenic reduction by Escherichia coli

    SciTech Connect

    Lahiri, Surobhi; Pulakat, Lakshmi; Gavini, Nara

    2008-04-04

    The NifH (dimer) and ArsA proteins are structural homologs and share common motifs like nucleotide-binding domains, signal transduction domains and also possible similar metal center ligands. Given the similarity between two proteins, we investigated if the NifH protein from Azotobacter vinelandii could functionally substitute for the ArsA1 half of the ArsA protein of Escherichia coli. The chimeric NifH-ArsA2 protein was expressed and detected in the E. coli strain by Western blotting. Growth comparisons of E. coli strains containing plasmids encoding for complete ArsA, partial ArsA (ArsA2) or chimeric ArsA (NifH-ArsA2) in media with increasing sodium arsenite concentrations (0-5 mM) showed that the chimeric NifH-ArsA2 could substitute for the ArsA. This functional complementation demonstrated the strong conservation of essential domains that have been maintained in NifH and ArsA even after their divergence to perform varied functions.

  17. Recombination–deletion between homologous cassettes in retrovirus is suppressed via a strategy of degenerate codon substitution

    PubMed Central

    Im, Eung Jun; Bais, Anthony J; Yang, Wen; Ma, Qiangzhong; Guo, Xiuyang; Sepe, Steven M; Junghans, Richard P

    2014-01-01

    Transduction and expression procedures in gene therapy protocols may optimally transfer more than a single gene to correct a defect and/or transmit new functions to recipient cells or organisms. This may be accomplished by transduction with two (or more) vectors, or, more efficiently, in a single vector. Occasionally, it may be useful to coexpress homologous genes or chimeric proteins with regions of shared homology. Retroviridae include the dominant vector systems for gene transfer (e.g., gamma-retro and lentiviruses) and are capable of such multigene expression. However, these same viruses are known for efficient recombination–deletion when domains are duplicated within the viral genome. This problem can be averted by resorting to two-vector strategies (two-chain two-vector), but at a penalty to cost, convenience, and efficiency. Employing a chimeric antigen receptor system as an example, we confirm that coexpression of two genes with homologous domains in a single gamma-retroviral vector (two-chain single-vector) leads to recombination–deletion between repeated sequences, excising the equivalent of one of the chimeric antigen receptors. Here, we show that a degenerate codon substitution strategy in the two-chain single-vector format efficiently suppressed intravector deletional loss with rescue of balanced gene coexpression by minimizing sequence homology between repeated domains and preserving the final protein sequence. PMID:25419532

  18. Stable mixed chimerism and tolerance to human organ transplants.

    PubMed

    Strober, Samuel

    2015-04-03

    Tolerance to combined kidney and hematopoietic cell transplant has been achieved in humans after establishment of mixed chimerism allowing for the withdrawal of immunosuppressive drugs. The seminal contributions of Ray Owen provided the scientific basis for the human protocol.

  19. Chimeric alignment by dynamic programming: Algorithm and biological uses

    SciTech Connect

    Komatsoulis, G.A.; Waterman, M.S.

    1997-12-01

    A new nearest-neighbor method for detecting chimeric 16S rRNA artifacts generated during PCR amplification from mixed populations has been developed. The method uses dynamic programming to generate an optimal chimeric alignment, defined as the highest scoring alignment between a query and a concatenation of a 5{prime} and a 3{prime} segment from two separate entries from a database of related sequences. Chimeras are detected by studying the scores and form of the chimeric and global sequence alignments. The chimeric alignment method was found to be marginally more effective than k-tuple based nearest-neighbor methods in simulation studies, but its most effective use is in concert with k-tuple methods. 15 refs., 3 figs., 1 tab.

  20. Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice.

    PubMed

    Tateno, Chise; Kawase, Yosuke; Tobita, Yoshimi; Hamamura, Satoko; Ohshita, Hiroki; Yokomichi, Hiroshi; Sanada, Harumi; Kakuni, Masakazu; Shiota, Akira; Kojima, Yuha; Ishida, Yuji; Shitara, Hiroshi; Wada, Naoko A; Tateishi, Hiromi; Sudoh, Masayuki; Nagatsuka, Shin-Ichiro; Jishage, Kou-Ichi; Kohara, Michinori

    2015-01-01

    We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression-not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for

  1. Chimeric mitochondrial peptides from contiguous regular and swinger RNA.

    PubMed

    Seligmann, Hervé

    2016-01-01

    Previous mass spectrometry analyses described human mitochondrial peptides entirely translated from swinger RNAs, RNAs where polymerization systematically exchanged nucleotides. Exchanges follow one among 23 bijective transformation rules, nine symmetric exchanges (X ↔ Y, e.g. A ↔ C) and fourteen asymmetric exchanges (X → Y → Z → X, e.g. A → C → G → A), multiplying by 24 DNA's protein coding potential. Abrupt switches from regular to swinger polymerization produce chimeric RNAs. Here, human mitochondrial proteomic analyses assuming abrupt switches between regular and swinger transcriptions, detect chimeric peptides, encoded by part regular, part swinger RNA. Contiguous regular- and swinger-encoded residues within single peptides are stronger evidence for translation of swinger RNA than previously detected, entirely swinger-encoded peptides: regular parts are positive controls matched with contiguous swinger parts, increasing confidence in results. Chimeric peptides are 200 × rarer than swinger peptides (3/100,000 versus 6/1000). Among 186 peptides with > 8 residues for each regular and swinger parts, regular parts of eleven chimeric peptides correspond to six among the thirteen recognized, mitochondrial protein-coding genes. Chimeric peptides matching partly regular proteins are rarer and less expressed than chimeric peptides matching non-coding sequences, suggesting targeted degradation of misfolded proteins. Present results strengthen hypotheses that the short mitogenome encodes far more proteins than hitherto assumed. Entirely swinger-encoded proteins could exist.

  2. Chimeric CYP21A1P/CYP21A2 genes identified in Czech patients with congenital adrenal hyperplasia.

    PubMed

    Vrzalová, Zuzana; Hrubá, Zuzana; Hrabincová, Eva Sťahlová; Vrábelová, Slávka; Votava, Felix; Koloušková, Stanislava; Fajkusová, Lenka

    2011-01-01

    Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders caused by an enzymatic deficiency which impairs the biosynthesis of cortisol and, in the majority of severe cases, also the biosynthesis of aldosterone. Approximately 95% of all CAH cases are caused by mutations in the steroid 21-hydroxylase gene (CYP21A2). The CYP21A2 gene and its inactive pseudogene (CYP21A1P) are located within the HLA class III region of the major histocompatibility complex (MHC) locus on chromosome 6p21.3. In this study, we describe chimeric CYP21A1P/CYP21A2 genes detected in our patients with 21-hydroxylase deficiency (21OHD). Chimeric CYP21A1P/CYP21A2 genes were present in 171 out of 508 mutated CYP21A2 alleles (33.8%). We detected four types of chimeric CYP21A1P/CYP21A2 genes: three of them have been described previously as CH-1, CH-3, CH-4, and one type is novel. The novel chimeric gene, termed CH-7, was detected in 21.4% of the mutant alleles. Possible causes of CYP21A1P/CYP21A2 formation are associated with 1) high recombination rate in the MHC locus, 2) high recombination rate between highly homologous genes and pseudogenes in the CYP21 gene area, and 3) the existence of chi-like sequences and repetitive minisatellite consensus sequences in CYP21A2 and CYP21A1P which play a role in promoting genetic recombination.

  3. Chimeric Antigen Receptor Therapy for Cancer

    PubMed Central

    Barrett, David M.; Singh, Nathan; Porter, David L.; Grupp, Stephan A.; June, Carl H.

    2014-01-01

    Improved outcomes for patients with cancer hinge on the development of new targeted therapies with acceptable short-term and long-term toxicity. Progress in basic, preclinical, and clinical arenas spanning cellular immunology, synthetic biology, and cell-processing technologies has paved the way for clinical applications of chimeric antigen receptor– based therapies. This new form of targeted immunotherapy merges the exquisite targeting specificity of monoclonal antibodies with the potent cytotoxicity and long-term persistence provided by cytotoxic T cells. Although this field is still in its infancy, clinical trials have already shown clinically significant antitumor activity in neuroblastoma, chronic lymphocytic leukemia, and B cell lymphoma, and trials targeting a variety of other adult and pediatric malignancies are under way. Ongoing work is focused on identifying optimal tumor targets and on elucidating and manipulating both cell- and host-associated factors to support expansion and persistence of the genetically engineered cells in vivo. The potential to target essentially any tumor-associated cell-surface antigen for which a monoclonal antibody can be made opens up an entirely new arena for targeted therapy of cancer. PMID:24274181

  4. Generating chimeric zebrafish embryos by transplantation.

    PubMed

    Kemp, Hilary A; Carmany-Rampey, Amanda; Moens, Cecilia

    2009-07-17

    One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

  5. Structure aided design of chimeric antibiotics.

    PubMed

    Karoli, Tomislav; Mamidyala, Sreeman K; Zuegg, Johannes; Fry, Scott R; Tee, Ernest H L; Bradford, Tanya A; Madala, Praveen K; Huang, Johnny X; Ramu, Soumya; Butler, Mark S; Cooper, Matthew A

    2012-04-01

    The rise of antibiotic resistance is of great clinical concern. One approach to reducing the development of resistance is to co-administer two or more antibiotics with different modes of action. However, it can be difficult to control the distribution and pharmacokinetics of two drugs to ensure both concentrations remain within the range of therapeutic efficacy whilst avoiding adverse effects. Hybrid drugs, where two drugs are linked together with a flexible linker, have been explored, but the resultant large, flexible molecules can have poor bioavailability. We have developed a chimeric approach using click chemistry where the pharmacophores of two drugs are overlapped into a single smaller, more drug-like molecule. Design and selection of compounds were assisted by in silico structural docking. We prepared a series of compounds that include candidates showing activity against the targets of both trimethoprim; dihydrofolate reductase, and ciprofloxacin; DNA gyrase and topoisomerase IV. The resultant triazole containing molecules show modest, but broad spectrum activities against drug sensitive and resistant Gram-negative and Gram-positive bacteria, with no observable cytotoxicity.

  6. Syngeneic Transplants with Modified Chimeric Hematopoietic Tumors.

    PubMed

    Hemann, Michael

    2015-08-03

    This protocol describes strategies to rapidly transduce tumor cells ex vivo and then transplant modified cells into immunocompetent-recipient mice. Inherent in the definition of a bona fide murine hematopoietic malignancy, unlike a myelo- or lympho-proliferative disease, is the ability to transplant tumors and give rise to a malignancy in recipient animals. This characteristic of hematopoietic disease makes these tumors a tractable model for examining the role of specific genes in tumor growth, dissemination, or therapeutic response. Additionally, because of the systemic nature of hematopoietic malignancies, transplanted tumors are frequently pathologically indistinguishable from donor malignancies-allowing one to perform decisive therapy studies on large cohorts of transplant recipients. Finally, following ex vivo manipulation, transplanted tumors can be made chimeric for the presence of defined retrovirally induced alterations. Thus, these malignancies can be made to resemble genetically heterogeneous human tumors that are in the process of acquiring new capabilities. In these experiments, fluorescent markers serve as a surrogate marker for the expression of a defined alteration, and the change in the percentage of fluorescent cells in a tumor population over time or in response to therapy can be used to gauge the impact of specific alterations on tumor behavior.

  7. 4-1BB chimeric antigen receptors.

    PubMed

    Campana, Dario; Schwarz, Herbert; Imai, Chihaya

    2014-01-01

    In addition to T-cell receptor signals, T lymphocytes require costimulatory signals for robust activation. Among these, those mediated by 4-1BB (CD137, TNFRSF9) are critical for tumor immunity. 4-1BB is expressed in T-cell receptor-activated lymphocytes as well as natural killer cells and other hematopoietic and nonhematopoietic cells. 4-1BB ligation induces a signaling cascade that results in cytokine production, expression of antiapoptotic molecules, and enhanced immune responses. In line with the described function of 4-1BB, its addition to CD3ζ chimeric antigen receptors (CARs) increases their capacity to provoke T-cell expansion and antitumor activity. The results of preclinical studies with 4-1BB CARs have been corroborated by encouraging results from clinical trials. Advantages and disadvantages of 4-1BB CARs versus CARs bearing other costimulatory components remain to be fully elucidated. In this review, we discuss the properties of 4-1BB, the design of 4-1BB CARs, and the function of T lymphocytes and natural killer cells expressing them.

  8. Chimeric human/murine monoclonal IgM antibodies to HIV-1 Nef antigen expressed on chronically infected cells.

    PubMed

    Kawai, Masahiro; He, Lianying; Kawamura, Takeshi; Omoto, Shinya; Fujii, Yoichi R; Okada, Noriko

    2003-01-01

    Human IgM antibody (Ab) to gangliosides induced cytolysis of HIV-1-infected cells by homologous human complement. We expected that any human IgM Ab reactive with HIV-1 infected cells could cause complement-mediated cytolysis. The trans-chromosome mouse (TC mouse) contains human chromosomes harboring genes responsible for immunoglobulin production. Spleen cells from TC mice immunized with recombinant Nef were fused with mouse myeloma cells to generate hybridomas, and we selected those that produced human mu-chain-positive Abs reactive with Nef fixed on an ELISA plate. However, the L-chain of the monoclonal Abs (mAbs) were murine lambda in type and were chimeric, and we could not succeed in obtaining mAb with human mu- and human kappa-chains. The chimeric mAbs reacted with the HIV-1 infected cells as seen with flow cytometric analysis, and the surface expression of Nef was also detectable on chronically infected OM10.1 cells which had no detectable gp120. However, although the reaction of the chimeric IgM mAb with HIV-1-infected MOLT4 cells induced C3 deposition on cell surfaces on incubation with fresh human serum, the cells remained unlysed, as determined by 51Cr release assay. The amount of Nef antigen on the cells might not have been high enough to overcome the function of HRF20 (CD59) that restricts formation of membrane attack complexes of homologous complement. However, combination of anti-Nef IgM mAb with other IgM mAbs reactive with the surface of HIV-1-infected cells may induce a synergistic effect in complement mediated cytolysis.

  9. Quantifying homologous replacement of loci between haloarchaeal species.

    PubMed

    Williams, David; Gogarten, J Peter; Papke, R Thane

    2012-01-01

    In vitro studies of the haloarchaeal genus Haloferax have demonstrated their ability to frequently exchange DNA between species, whereas rates of homologous recombination estimated from natural populations in the genus Halorubrum are high enough to maintain random association of alleles between five loci. To quantify the effects of gene transfer and recombination of commonly held (relaxed core) genes during the evolution of the class Halobacteria (haloarchaea), we reconstructed the history of 21 genomes representing all major groups. Using a novel algorithm and a concatenated ribosomal protein phylogeny as a reference, we created a directed horizontal genetic transfer (HGT) network of contemporary and ancestral genomes. Gene order analysis revealed that 90% of testable HGTs were by direct homologous replacement, rather than nonhomologous integration followed by a loss. Network analysis revealed an inverse log-linear relationship between HGT frequency and ribosomal protein evolutionary distance that is maintained across the deepest divergences in Halobacteria. We use this mathematical relationship to estimate the total transfers and amino acid substitutions delivered by HGTs in each genome, providing a measure of chimerism. For the relaxed core genes of each genome, we conservatively estimate that 11-20% of their evolution occurred in other haloarchaea. Our findings are unexpected, because the transfer and homologous recombination of relaxed core genes between members of the class Halobacteria disrupts the coevolution of genes; however, the generation of new combinations of divergent but functionally related genes may lead to adaptive phenotypes not available through cumulative mutations and recombination within a single population.

  10. Quantifying Homologous Replacement of Loci between Haloarchaeal Species

    PubMed Central

    Williams, David; Gogarten, J. Peter; Papke, R. Thane

    2012-01-01

    In vitro studies of the haloarchaeal genus Haloferax have demonstrated their ability to frequently exchange DNA between species, whereas rates of homologous recombination estimated from natural populations in the genus Halorubrum are high enough to maintain random association of alleles between five loci. To quantify the effects of gene transfer and recombination of commonly held (relaxed core) genes during the evolution of the class Halobacteria (haloarchaea), we reconstructed the history of 21 genomes representing all major groups. Using a novel algorithm and a concatenated ribosomal protein phylogeny as a reference, we created a directed horizontal genetic transfer (HGT) network of contemporary and ancestral genomes. Gene order analysis revealed that 90% of testable HGTs were by direct homologous replacement, rather than nonhomologous integration followed by a loss. Network analysis revealed an inverse log-linear relationship between HGT frequency and ribosomal protein evolutionary distance that is maintained across the deepest divergences in Halobacteria. We use this mathematical relationship to estimate the total transfers and amino acid substitutions delivered by HGTs in each genome, providing a measure of chimerism. For the relaxed core genes of each genome, we conservatively estimate that 11–20% of their evolution occurred in other haloarchaea. Our findings are unexpected, because the transfer and homologous recombination of relaxed core genes between members of the class Halobacteria disrupts the coevolution of genes; however, the generation of new combinations of divergent but functionally related genes may lead to adaptive phenotypes not available through cumulative mutations and recombination within a single population. PMID:23160063

  11. Engineered Zinc Finger Nuclease–Mediated Homologous Recombination of the Human Rhodopsin Gene

    PubMed Central

    Greenwald, David L.; Cashman, Siobhan M.

    2010-01-01

    Purpose. Novel zinc finger nucleases (ZFNs) were designed to target the human rhodopsin gene and induce homologous recombination of a donor DNA fragment. Methods. Three-finger zinc finger nucleases were designed based on previously published guidelines. To assay for ZFN specificity, the authors generated human embryonic retinoblast cell lines stably expressing a Pro23His rhodopsin, the most common mutation associated with autosomal dominant retinitis pigmentosa in North America. They report quantification of these rhodopsin-specific ZFNs to induce a targeted double-strand break in the human genome, demonstrate their ability to induce homologous recombination of a donor DNA fragment, and report the quantification of the frequency of ZFN-mediated homologous recombination. Results. Compared with endogenous homologous recombination, the authors observed a 12-fold increase in homologous recombination and an absolute frequency of ZFN-directed homologous recombination as high as 17% in the human rhodopsin gene. Conclusions. ZFNs are chimeric proteins with significant potential for the treatment of inherited diseases. In this study, the authors report the design of novel ZFNs targeting the human rhodopsin gene. These ZFNs may be useful for the treatment of retinal diseases such as retinitis pigmentosa, one of the most common causes of inherited blindness in the developed world. Herein, they also report on several aspects of donor fragment design and in vitro conditions that facilitate ZFN-mediated homologous recombination. PMID:20671268

  12. Engineered zinc finger nuclease-mediated homologous recombination of the human rhodopsin gene.

    PubMed

    Greenwald, David L; Cashman, Siobhan M; Kumar-Singh, Rajendra

    2010-12-01

    Novel zinc finger nucleases (ZFNs) were designed to target the human rhodopsin gene and induce homologous recombination of a donor DNA fragment. Three-finger zinc finger nucleases were designed based on previously published guidelines. To assay for ZFN specificity, the authors generated human embryonic retinoblast cell lines stably expressing a Pro23His rhodopsin, the most common mutation associated with autosomal dominant retinitis pigmentosa in North America. They report quantification of these rhodopsin-specific ZFNs to induce a targeted double-strand break in the human genome, demonstrate their ability to induce homologous recombination of a donor DNA fragment, and report the quantification of the frequency of ZFN-mediated homologous recombination. Compared with endogenous homologous recombination, the authors observed a 12-fold increase in homologous recombination and an absolute frequency of ZFN-directed homologous recombination as high as 17% in the human rhodopsin gene. ZFNs are chimeric proteins with significant potential for the treatment of inherited diseases. In this study, the authors report the design of novel ZFNs targeting the human rhodopsin gene. These ZFNs may be useful for the treatment of retinal diseases such as retinitis pigmentosa, one of the most common causes of inherited blindness in the developed world. Herein, they also report on several aspects of donor fragment design and in vitro conditions that facilitate ZFN-mediated homologous recombination.

  13. The Chimeric Protein Domain III-Capsid of Dengue Virus Serotype 2 (DEN-2) Successfully Boosts Neutralizing Antibodies Generated in Monkeys upon Infection with DEN-2▿

    PubMed Central

    Valdés, Iris; Gil, Lázaro; Romero, Yaremis; Castro, Jorge; Puente, Pedro; Lazo, Laura; Marcos, Ernesto; Guzmán, María G.; Guillén, Gerardo; Hermida, Lisset

    2011-01-01

    Use of a heterologous prime-boost strategy based on a combination of nonreplicative immunogens and candidate attenuated virus vaccines against dengue virus in the same schedule is an attractive approach. These combinations may result in a condensed immunization regime for humans, thus reducing the number of doses with attenuated virus and the time spacing. The present work deals with the evaluation of the heterologous prime-boost strategy combining a novel chimeric protein (domain III-capsid) of dengue virus serotype 2 (DEN-2) and the infective homologous virus in the same immunization schedule in monkeys. Primed monkeys received one dose of infective DEN-2 and were then vaccinated with the recombinant protein. We found that animals developed a neutralizing antibody response after the infective dose and were notably boosted with a second dose of the chimeric protein 3 months later. The neutralizing antibodies induced were long lasting, and animals also showed the ability to induce a specific cellular response 6 months after the booster dose. As a conclusion, we can state that the domain III region, when it is properly presented as a fusion protein to the immune system, is able to recall the neutralizing antibody response elicited following homologous virus infection in monkeys. Further prime-boost approaches can be performed in a condensed regime combining the chimeric domain III-capsid protein and candidate live attenuated vaccines against DEN-2. PMID:21209159

  14. Field homology: a meaningful definition.

    PubMed

    Cookson, K

    2001-02-01

    Field homology refers to populations of cells that derive from evolutionarily conserved regions of embryos but are distributed across sets of adult morphological structures that cannot be placed in one-to-one correspondance. The concept of field homology has proven especially attractive to comparative neurologists because it allows them to deal with the fact that sets of nuclei or nuclear subdivisions often cannot be compared on a one-to-one basis across phyletic groups. However, the concept of field homology has recently come under criticism. It has been argued that field homology is theoretically impossible because it requires sequences of developmental stages to be both evolutionarily conserved and evolutionarily modified. It has also been argued that field homology allows overly vague comparisons of adult morphological structures, fails to account for homologous structures that derive from non-homologous embryonic sources, and establishes overly rigid links between embryonic and adult morphology. All of these criticisms may be adequately addressed by explaining field homology in terms of differentiation. The present paper explains field homology in terms of differentiation using the amniote dorsal thalamus to illustrate major points. It is concluded that field homology is a meaningful concept when defined in terms of differentiation, applied to appropriate cases, and properly limited in its comparisons of adult structures.

  15. Rhythmical bimanual force production: homologous and non-homologous muscles.

    PubMed

    Kennedy, Deanna M; Boyle, Jason B; Rhee, Joohyun; Shea, Charles H

    2015-01-01

    The experiment was designed to determine participants' ability to coordinate a bimanual multifrequency pattern of isometric forces using homologous or non-homologous muscles. Lissajous feedback was provided to reduce perceptual and attentional constraints. The primary purpose was to determine whether the activation of homologous and non-homologous muscles resulted in different patterns of distortions in the left limb forces that are related to the forces produced by the right limb. The task was to rhythmically produce a 1:2 pattern of isometric forces by exerting isometric forces on the left side force transducer with the left arm that was coordinated with the pattern of isometric forces produced on the right side force transducer with the right arm. The results indicated that participants were able to 'tune-in' a 1:2 coordination patterns using homologous (triceps muscles of the left and right limbs) and using non-homologous muscles (biceps left limb and triceps right limb) when provided Lissajous feedback. However, distinct but consistent and identifiable distortions in the left limb force traces were observed for both the homologous and non-homologous tasks. For the homologous task, the interference occurred in the left limb when the right limb was initiating and releasing force. For the non-homologous task, the interference in the left limb force occurred only when the right limb was releasing force. In both conditions, the interference appeared to continue from the point of force initiation and/or release to peak force velocity. The overall results are consistent with the notion that neural crosstalk manifests differently during the coordination of the limbs depending upon whether homologous or non-homologous muscles are activated.

  16. Homology, homoplasy, novelty, and behavior.

    PubMed

    Hall, Brian K

    2013-01-01

    Richard Owen coined the modern definition of homology in 1843. Owen's conception of homology was pre-evolutionary, nontransformative (homology maintained basic plans or archetypes), and applied to the fully formed structures of animals. I sketch out the transition to an evolutionary approach to homology in which all classes of similarity are interpreted against the single branching tree of life, and outline the evidence for the application of homology across all levels and features of the biological hierarchy, including behavior. Owen contrasted homology with analogy. While this is not incorrect it is a pre-evolutionary contrast. Lankester [Lankester [1870] Journal of Natural History, 6 (31), 34-43] proposed homoplasy as the class of homology applicable to features formed by independent evolution. Today we identify homology, convergence, parallelism, and novelties as patterns of evolutionary change. A central issue in homology [Owen [1843] Lectures on comparative anatomy and physiology of the invertebrate animals, delivered at the Royal College of Surgeons in 1843. London: Longman, Brown, Green & Longmans] has been whether homology of features-the "same" portion of the brain in different species, for example-depends upon those features sharing common developmental pathways. Owen did not require this criterion, although he observed that homologues often do share developmental pathways (and we now know, often share gene pathways). A similar situation has been explored in the study of behavior, especially whether behaviors must share a common structural, developmental, neural, or genetic basis to be classified as homologous. However, and importantly, development and genes evolve. As shown with both theory and examples, morphological and behavioral features of the phenotype can be homologized as structural or behavioral homologues, respectively, even when their developmental or genetic bases differ (are not homologous). Copyright © 2012 Wiley Periodicals, Inc.

  17. Immunogenicity and efficacy of chimeric dengue vaccine (DENVax) formulations in interferon-deficient AG129 mice.

    PubMed

    Brewoo, Joseph N; Kinney, Richard M; Powell, Tim D; Arguello, John J; Silengo, Shawn J; Partidos, Charalambos D; Huang, Claire Y-H; Stinchcomb, Dan T; Osorio, Jorge E

    2012-02-14

    Formulations of chimeric dengue vaccine (DENVax) viruses containing the pre-membrane (prM) and envelope (E) genes of serotypes 1-4 expressed in the context of the attenuated DENV-2 PDK-53 genome were tested for safety, immunogenicity and efficacy in interferon receptor knock-out mice (AG129). Monovalent formulations were safe and elicited robust neutralizing antibody responses to the homologous virus and only limited cross-reactivity to other serotypes. A single dose of monovalent DENVax-1, -2, or -3 vaccine provided eighty or greater percent protection against both wild-type (wt) DENV-1 (Mochizuki strain) and DENV-2 (New Guinea C strain) challenge viruses. A single dose of monovalent DENVax-4 also provided complete protection against wt DENV-1 challenge and significantly increased the survival times after challenge with wt DENV-2. In studies using tetravalent mixtures, DENVax ratios were identified that: (i) caused limited viremia, (ii) induced serotype-specific neutralizing antibodies to all four DENV serotypes with different hierarchies, and (iii) conferred full protection against clinical signs of disease following challenge with either wt DENV-1 or DENV-2 viruses. Overall, these data highlight the immunogenic profile of DENVax, a novel candidate tetravalent dengue vaccine and the advantage of sharing a common attenuated genomic backbone among the DENVax monovalent vaccines that confer protection against homologous or heterologous virus challenge.

  18. Steroid metabolism in chimeric mice with humanized liver.

    PubMed

    Lootens, Leen; Van Eenoo, Peter; Meuleman, Philip; Pozo, Oscar J; Van Renterghem, Pieter; Leroux-Roels, Geert; Delbeke, Frans T

    2009-11-01

    Anabolic androgenic steroids are considered to be doping agents and are prohibited in sports. Their metabolism needs to be elucidated to allow for urinary detection by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Steroid metabolism was assessed using uPA(+/+) SCID mice with humanized livers (chimeric mice). This study presents the results of 19-norandrost-4-ene-3,17-dione (19-norAD) administration to these in vivo mice. As in humans, 19-norandrosterone and 19-noretiocholanolone are the major detectable metabolites of 19-norAD in the urine of chimeric mice.A summary is given of the metabolic pathways found in chimeric mice after administration of three model steroid compounds (methandienone, androst-4-ene-3,17-dione and 19-norandrost-4-ene-3,17-dione). From these studies we can conclude that all major metabolic pathways for anabolic steroids in humans are present in the chimeric mouse. It is hoped that, in future, this promising chimeric mouse model might assist the discovery of new and possible longer detectable metabolites of (designer) steroids.

  19. Assessment of chimerism in epithelial cancers in transplanted patients.

    PubMed

    Leboeuf, Christophe; Ratajczak, Philippe; Vérine, Jérôme; Elbouchtaoui, Morad; Plassa, François; Legrès, Luc; Ferreira, Irmine; Sandid, Wissam; Varna, Mariana; Bousquet, Guilhem; Verneuil, Laurence; Janin, Anne

    2014-01-01

    Cancer is now the most severe complication in the long term in transplant recipients. As most solid-organ or hematopoietic stem-cell transplantations are allogeneic, chimerism studies can be performed on cancers occurring in recipients. We summarize here the different methods used to study chimerism in cancers developing in allogeneic-transplant recipients, analyze their respective advantages and report the main results obtained from these studies. Chimerism analyses of cancers in transplant recipients require methods suited to tissue samples. In the case of gender-mismatched transplantation, the XY chromosomes can be explored using fluorescent in situ hybridization on whole-tissue sections or Y-sequence-specific PCR after the laser microdissection of tumor cells. For cancers occurring after gender-matched transplantation, laser microdissection of tumor cells enables studies of microsatellite markers and high-resolution melting analysis of mitochondrial DNA on genes with marked polymorphism, provided these are different in the donor and the recipient. The results of different studies address the cancers that develop in both recipients and in transplants. The presence of chimeric cells in these two types of cancer implies an exchange of progenitor/stem-cells between transplant and recipient, and the plasticity of these progenitor/stem-cells contributes to epithelial cancers. The presence of chimeric cells in concomitant cancers and preneoplastic lesions implies that the oncogenesis of these cancers progresses through a multistep process.

  20. Preclinical Model To Test Human Papillomavirus Virus (HPV) Capsid Vaccines In Vivo Using Infectious HPV/Cottontail Rabbit Papillomavirus Chimeric Papillomavirus Particles▿

    PubMed Central

    Mejia, Andres F.; Culp, Timothy D.; Cladel, Nancy M.; Balogh, Karla K.; Budgeon, Lynn R.; Buck, Christopher B.; Christensen, Neil D.

    2006-01-01

    A human papillomavirus (HPV) vaccine consisting of virus-like particles (VLPs) was recently approved for human use. It is generally assumed that VLP vaccines protect by inducing type-specific neutralizing antibodies. Preclinical animal models cannot be used to test for protection against HPV infections due to species restriction. We developed a model using chimeric HPV capsid/cottontail rabbit papillomavirus (CRPV) genome particles to permit the direct testing of HPV VLP vaccines in rabbits. Animals vaccinated with CRPV, HPV type 16 (HPV-16), or HPV-11 VLPs were challenged with both homologous (CRPV capsid) and chimeric (HPV-16 capsid) particles. Strong type-specific protection was observed, demonstrating the potential application of this approach. PMID:17005666

  1. Homological Computation Using Spanning Trees

    NASA Astrophysics Data System (ADS)

    Molina-Abril, H.; Real, P.

    We introduce here a new mathbb{F}_2 homology computation algorithm based on a generalization of the spanning tree technique on a finite 3-dimensional cell complex K embedded in ℝ3. We demonstrate that the complexity of this algorithm is linear in the number of cells. In fact, this process computes an algebraic map φ over K, called homology gradient vector field (HGVF), from which it is possible to infer in a straightforward manner homological information like Euler characteristic, relative homology groups, representative cycles for homology generators, topological skeletons, Reeb graphs, cohomology algebra, higher (co)homology operations, etc. This process can be generalized to others coefficients, including the integers, and to higher dimension.

  2. Vaccine Efficacy of Inactivated, Chimeric Hemagglutinin H9/H5N2 Avian Influenza Virus and Its Suitability for the Marker Vaccine Strategy.

    PubMed

    Kim, Se Mi; Kim, Young-Il; Park, Su-Jin; Kim, Eun-Ha; Kwon, Hyeok-Il; Si, Young-Jae; Lee, In-Won; Song, Min-Suk; Choi, Young Ki

    2017-03-15

    In order to produce a dually effective vaccine against H9 and H5 avian influenza viruses that aligns with the DIVA (differentiating infected from vaccinated animals) strategy, we generated a chimeric H9/H5N2 recombinant vaccine that expressed the whole HA1 region of A/CK/Korea/04163/04 (H9N2) and the HA2 region of recent highly pathogenic avian influenza (HPAI) A/MD/Korea/W452/14 (H5N8) viruses. The chimeric H9/H5N2 virus showed in vitro and in vivo growth properties and virulence that were similar to those of the low-pathogenic avian influenza (LPAI) H9 virus. An inactivated vaccine based on this chimeric virus induced serum neutralizing (SN) antibodies against both H9 and H5 viruses but induced cross-reactive hemagglutination inhibition (HI) antibody only against H9 viruses. Thus, this suggests its compatibility for use in the DIVA strategy against H5 strains. Furthermore, the chimeric H9/H5N2 recombinant vaccine protected immunized chickens against lethal challenge by HPAI H5N8 viruses and significantly attenuated virus shedding after infection by both H9N2 and HPAI H5N8 viruses. In mice, serological analyses confirmed that HA1- and HA2 stalk-specific antibody responses were induced by vaccination and that the DIVA principle could be employed through the use of an HI assay against H5 viruses. Furthermore, each HA1- and HA2 stalk-specific antibody response was sufficient to inhibit viral replication and protect the chimeric virus-immunized mice from lethal challenge with both mouse-adapted H9N2 and wild-type HPAI H5N1 viruses, although differences in vaccine efficacy against a homologous H9 virus (HA1 head domain immune-mediated protection) and a heterosubtypic H5 virus (HA2 stalk domain immune-mediated protection) were observed. Taken together, these results demonstrate that the novel chimeric H9/H5N2 recombinant virus is a low-pathogenic virus, and this chimeric vaccine is suitable for a DIVA vaccine with broad-spectrum neutralizing antibody against H5 avian

  3. Facilitating cells: Translation of hematopoietic chimerism to achieve clinical tolerance

    PubMed Central

    Ildstad, Suzanne T.; Leventhal, Joseph; Wen, Yujie; Yolcu, Esma

    2015-01-01

    ABSTRACT For over 50 y the association between hematopoietic chimerism and tolerance has been recognized. This originated with the brilliant observation by Dr. Ray Owen that freemartin cattle twins that shared a common placental blood supply were red blood cell chimeras, which led to the discovery that hematopoietic chimerism resulted in actively acquired tolerance. This was first confirmed in neonatal mice by Medawar et al. and subsequently in adult rodents. Fifty years later this concept has been successfully translated to solid organ transplant recipients in the clinic. The field is new, but cell-based therapies are being used with increasing frequency to induce tolerance and immunomodulation. The future is bright. This review focuses on chimerism and tolerance: past, present and prospects for the future. PMID:26745761

  4. A chimeric toxin vaccine protects against primary and recurrent Clostridium difficile infection.

    PubMed

    Wang, Haiying; Sun, Xingmin; Zhang, Yongrong; Li, Shan; Chen, Kevin; Shi, Lianfa; Nie, Weijia; Kumar, Raj; Tzipori, Saul; Wang, Jufang; Savidge, Tor; Feng, Hanping

    2012-08-01

    The global emergence of Clostridium difficile infection (CDI) has contributed to the recent surge in severe antibiotic-associated diarrhea and colonic inflammation. C. difficile produces two homologous glucosylating exotoxins, TcdA and TcdB, both of which are pathogenic and require neutralization to prevent disease occurrence. However, because of their large size and complex multifunctional domain structures, it has been a challenge to produce native recombinant toxins that may serve as vaccine candidates. Here, we describe a novel chimeric toxin vaccine that retains major neutralizing epitopes from both toxins and confers complete protection against primary and recurrent CDI in mice. Using a nonpathogenic Bacillus megaterium expression system, we generated glucosyltransferase-deficient holotoxins and demonstrated their loss of toxicity. The atoxic holotoxins induced potent antitoxin neutralizing antibodies showing little cross-immunogenicity or protection between TcdA and TcdB. To facilitate simultaneous protection against both toxins, we generated an active clostridial toxin chimera by switching the receptor binding domain of TcdB with that of TcdA. The toxin chimera was fully cytotoxic and showed potent proinflammatory activities. This toxicity was essentially abolished in a glucosyltransferase-deficient toxin chimera, cTxAB. Parenteral immunization of mice or hamsters with cTxAB induced rapid and potent neutralizing antibodies against both toxins. Complete and long-lasting disease protection was conferred by cTxAB vaccinations against both laboratory and hypervirulent C. difficile strains. Finally, prophylactic cTxAB vaccination prevented spore-induced disease relapse, which constitutes one of the most significant clinical issues in CDI. Thus, the rational design of recombinant chimeric toxins provides a novel approach for protecting individuals at high risk of developing CDI.

  5. Comparison of Recombinant Trypanosoma cruzi Peptide Mixtures versus Multiepitope Chimeric Proteins as Sensitizing Antigens for Immunodiagnosis▿

    PubMed Central

    Camussone, Cecilia; Gonzalez, Verónica; Belluzo, María S.; Pujato, Nazarena; Ribone, María E.; Lagier, Claudia M.; Marcipar, Iván S.

    2009-01-01

    The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The better-performing Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula +OD/cutoff value (where +OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp. PMID:19339486

  6. Comparison of recombinant Trypanosoma cruzi peptide mixtures versus multiepitope chimeric proteins as sensitizing antigens for immunodiagnosis.

    PubMed

    Camussone, Cecilia; Gonzalez, Verónica; Belluzo, María S; Pujato, Nazarena; Ribone, María E; Lagier, Claudia M; Marcipar, Iván S

    2009-06-01

    The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The better-performing Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula +OD/cutoff value (where +OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp.

  7. 78 FR 16505 - Prospective Grant of Exclusive License: Chimeric West Nile/Dengue Viruses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-15

    ...: Chimeric West Nile/Dengue Viruses AGENCY: Centers for Disease Control and Prevention (CDC), Department of... Application 61/049,342, filed 4/30/2008, entitled ``Engineered, Chimeric West Nile/Dengue Viruses;'' PCT..., filed 10/29/2010, entitled ``Chimeric West Nile/Dengue Viruses;'' and all related continuing and foreign...

  8. Evolving the Concept of Homology

    ERIC Educational Resources Information Center

    Naples, Virginia L.; Miller, Jon S.

    2009-01-01

    Understanding homology is fundamental to learning about evolution. The present study shows an exercise that can be varied in complexity, for which students compile research illustrating the fate of homologous fish skull elements, and assemble a mural to serve as a learning aid. The skull of the most primitive living Actinopterygian (bony fish),…

  9. Therapeutic use of chimeric bacteriophage (phage) lysins in staphylococcal endophthalmitis

    USDA-ARS?s Scientific Manuscript database

    Purpose: Phage endolysins are peptidoglycan hydrolases that are produced at the end of the phage lytic cycle to digest the host bacterial cell wall, facilitating the release of mature phage progeny. The aim of this study is to determine the antimicrobial activity of chimeric phage lysins against cli...

  10. Adaptive impact of the chimeric gene Quetzalcoatl in Drosophila melanogaster.

    PubMed

    Rogers, Rebekah L; Bedford, Trevor; Lyons, Ana M; Hartl, Daniel L

    2010-06-15

    Chimeric genes, which form through the genomic fusion of two protein-coding genes, are a significant source of evolutionary novelty in Drosophila melanogaster. However, the propensity of chimeric genes to produce adaptive phenotypic changes is not fully understood. Here, we describe the chimeric gene Quetzalcoatl (Qtzl; CG31864), which formed in the recent past and swept to fixation in D. melanogaster. Qtzl arose through a duplication on chromosome 2L that united a portion of the mitochondrially targeted peptide CG12264 with a segment of the polycomb gene escl. The 3' segment of the gene, which is derived from escl, is inherited out of frame, producing a unique peptide sequence. Nucleotide diversity is drastically reduced and site frequency spectra are significantly skewed surrounding the duplicated region, a finding consistent with a selective sweep on the duplicate region containing Qtzl. Qtzl has an expression profile that largely resembles that of escl, with expression in early pupae, adult females, and male testes. However, expression patterns appear to have been decoupled from both parental genes during later embryonic development and in head tissues of adult males, indicating that Qtzl has developed a distinct regulatory profile through the rearrangement of different 5' and 3' regulatory domains. Furthermore, misexpression of Qtzl suppresses defects in the formation of the neuromuscular junction in larvae, demonstrating that Qtzl can produce phenotypic effects in cells. Together, these results show that chimeric genes can produce structural and regulatory changes in a single mutational step and may be a major factor in adaptive evolution.

  11. Ray Owen and the history of naturally acquired chimerism

    PubMed Central

    Martin, Aryn

    2015-01-01

    abstract This article interweaves a history of Ray Owen's early work with a broader account of the conceptual landscape of immunology in the mid 1950's. In particular, Owen's openness to the very possibility of chimeric phenomena is recognized. PMID:27093621

  12. Systematic evaluation of atmospheric chemistry-transport model CHIMERE

    NASA Astrophysics Data System (ADS)

    Khvorostyanov, Dmitry; Menut, Laurent; Mailler, Sylvain; Siour, Guillaume; Couvidat, Florian; Bessagnet, Bertrand; Turquety, Solene

    2017-04-01

    Regional-scale atmospheric chemistry-transport models (CTM) are used to develop air quality regulatory measures, to support environmentally sensitive decisions in the industry, and to address variety of scientific questions involving the atmospheric composition. Model performance evaluation with measurement data is critical to understand their limits and the degree of confidence in model results. CHIMERE CTM (http://www.lmd.polytechnique.fr/chimere/) is a French national tool for operational forecast and decision support and is widely used in the international research community in various areas of atmospheric chemistry and physics, climate, and environment (http://www.lmd.polytechnique.fr/chimere/CW-articles.php). This work presents the model evaluation framework applied systematically to the new CHIMERE CTM versions in the course of the continuous model development. The framework uses three of the four CTM evaluation types identified by the Environmental Protection Agency (EPA) and the American Meteorological Society (AMS): operational, diagnostic, and dynamic. It allows to compare the overall model performance in subsequent model versions (operational evaluation), identify specific processes and/or model inputs that could be improved (diagnostic evaluation), and test the model sensitivity to the changes in air quality, such as emission reductions and meteorological events (dynamic evaluation). The observation datasets currently used for the evaluation are: EMEP (surface concentrations), AERONET (optical depths), and WOUDC (ozone sounding profiles). The framework is implemented as an automated processing chain and allows interactive exploration of the results via a web interface.

  13. Chimeric Genes in Deletions and Duplications Associated with Intellectual Disability

    PubMed Central

    Monfort, Sandra; Roselló, Mónica; Oltra, Silvestre; Caro-Llopis, Alfonso

    2017-01-01

    We report on three nonrelated patients with intellectual disability and CNVs that give rise to three new chimeric genes. All the genes forming these fusion transcripts may have an important role in central nervous system development and/or in gene expression regulation, and therefore not only their deletion or duplication but also the resulting chimeric gene may contribute to the phenotype of the patients. Deletions and duplications are usually pathogenic when affecting dose-sensitive genes. Alternatively, a chimeric gene may also be pathogenic by different gain-of-function mechanisms that are not restricted to dose-sensitive genes: the emergence of a new polypeptide that combines functional domains from two different genes, the deregulated expression of any coding sequence by the promoter region of a neighboring gene, and/or a putative dominant-negative effect due to the preservation of functional domains of partially truncated proteins. Fusion oncogenes are well known, but in other pathologies, the search for chimeric genes is disregarded. According to our findings, we hypothesize that the frequency of fusion transcripts may be much higher than suspected, and it should be taken into account in the array-CGH analyses of patients with intellectual disability. PMID:28630856

  14. Synthesis of a heterogeneous artificial metallolipase with chimeric catalytic activity.

    PubMed

    Filice, M; Romero, O; Gutiérrez-Fernández, J; de Las Rivas, B; Hermoso, J A; Palomo, J M

    2015-06-07

    A solid-phase strategy using lipase as a biomolecular scaffold to produce a large amount of Cu(2+)-metalloenzyme is proposed here. The application of this protocol on different 3D cavities of the enzyme allows creating a heterogeneous artificial metallolipase showing chimeric catalytic activity. The artificial catalyst was assessed in Diels-Alder cycloaddition reactions and cascade reactions showing excellent catalytic properties.

  15. Construction of yellow fever-influenza A chimeric virus particles.

    PubMed

    Oliveira, B C E P D; Liberto, M I M; Barth, O M; Cabral, M C

    2002-12-01

    In order to obtain a better understanding of the functional mechanisms involved in the fusogenesis of enveloped viruses, the influenza A (X31) and the yellow fever (17DD) virus particles were used to construct a chimeric structure based on their distinct pH requirements for fusion, and the distinct malleability of their nucleocapsids. The malleable nucleocapsid of the influenza A virus particle is characterized by a pleomorphic configuration when observed by electron microscopy. A heat inactivated preparation of X31 virus was used as a lectin to interact with the sialic acid domains present in the 17DD virus envelope. The E spikes of 17DD virus were induced to promote fusion of both envelopes, creating a double genome enveloped structure, the chimeric yellow fever-influenza A virus particle. These chimeric viral particles, originally denominated 'partículas virais quiméricas' (PVQ), were characterized by their infectious capacity for different biological systems. Cell inoculation with PVQ resulted in viral products that showed similar characteristics to those obtained after 17DD virus infections. Our findings open new opportunities towards the understanding of both virus particles and aspects of cellular physiologic quality control. The yellow fever-influenza A chimeric particles, by means of their hybrid composition, should be a valuable tool in the study of cell biology and the function of viral components.

  16. Chimeric aptamers in cancer cell-targeted drug delivery

    PubMed Central

    Kanwar, Jagat R; Roy, Kislay; Kanwar, Rupinder K

    2011-01-01

    Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets ("apatopes") with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern Pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer-aptamer, aptamer-nonaptamer biomacromolecules (siRNAs, proteins) and aptamer-nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities. PMID:21955150

  17. Chimeric L2-Based Virus-Like Particle (VLP) Vaccines Targeting Cutaneous Human Papillomaviruses (HPV)

    PubMed Central

    Huber, Bettina; Schellenbacher, Christina; Shafti-Keramat, Saeed; Jindra, Christoph; Christensen, Neil

    2017-01-01

    Common cutaneous human papillomavirus (HPV) types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP) self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas), but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa) 17–36) on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV) neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV. In vivo cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross-) protected against beta HPV5/20/24/38/96/16 (but not type 76), while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target cutaneous

  18. Chimeric L2-Based Virus-Like Particle (VLP) Vaccines Targeting Cutaneous Human Papillomaviruses (HPV).

    PubMed

    Huber, Bettina; Schellenbacher, Christina; Shafti-Keramat, Saeed; Jindra, Christoph; Christensen, Neil; Kirnbauer, Reinhard

    2017-01-01

    Common cutaneous human papillomavirus (HPV) types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP) self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas), but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa) 17-36) on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV) neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV. In vivo cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross-) protected against beta HPV5/20/24/38/96/16 (but not type 76), while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target cutaneous HPV

  19. Structural basis for drug and substrate specificity exhibited by FIV encoding a chimeric FIV/HIV protease

    PubMed Central

    Lin, Ying-Chuan; Perryman, Alexander L.; Olson, Arthur J.; Torbett, Bruce E.; Elder, John H.; Stout, C. David

    2011-01-01

    A chimeric feline immunodeficiency virus (FIV) protease (PR) has been engineered that supports infectivity but confers sensitivity to the human immunodeficiency virus (HIV) PR inhibitors darunavir (DRV) and lopinavir (LPV). The 6s-98S PR has five replacements mimicking homologous residues in HIV PR and a sixth which mutated from Pro to Ser during selection. Crystal structures of the 6s-98S FIV PR chimera with DRV and LPV bound have been determined at 1.7 and 1.8 Å resolution, respectively. The structures reveal the role of a flexible 90s loop and residue 98 in supporting Gag processing and infectivity and the roles of residue 37 in the active site and residues 55, 57 and 59 in the flap in conferring the ability to specifically recognize HIV PR drugs. Specifically, Ile37Val preserves tertiary structure but prevents steric clashes with DRV and LPV. Asn55Met and Val59Ile induce a distinct kink in the flap and a new hydrogen bond to DRV. Ile98Pro→Ser and Pro100Asn increase 90s loop flexibility, Gln99Val contributes hydrophobic contacts to DRV and LPV, and Pro100Asn forms compensatory hydrogen bonds. The chimeric PR exhibits a comparable number of hydrogen bonds, electrostatic interactions and hydrophobic contacts with DRV and LPV as in the corresponding HIV PR complexes, consistent with IC50 values in the nanomolar range. PMID:21636894

  20. Fivebranes and 3-manifold homology

    NASA Astrophysics Data System (ADS)

    Gukov, Sergei; Putrov, Pavel; Vafa, Cumrun

    2017-07-01

    Motivated by physical constructions of homological knot invariants, we study their analogs for closed 3-manifolds. We show that fivebrane compactifications provide a universal description of various old and new homological invariants of 3-manifolds. In terms of 3d/3d correspondence, such invariants are given by the Q-cohomology of the Hilbert space of partially topologically twisted 3d N=2 theory T[ M 3] on a Riemann surface with defects. We demonstrate this by concrete and explicit calculations in the case of monopole/Heegaard Floer homology and a 3-manifold analog of Khovanov-Rozansky link homology. The latter gives a categorification of Chern-Simons partition function. Some of the new key elements include the explicit form of the S-transform and a novel connection between categorification and a previously mysterious role of Eichler integrals in Chern-Simons theory.

  1. Abelian link invariants and homology

    SciTech Connect

    Guadagnini, Enore; Mancarella, Francesco

    2010-06-15

    We consider the link invariants defined by the quantum Chern-Simons field theory with compact gauge group U(1) in a closed oriented 3-manifold M. The relation of the Abelian link invariants with the homology group of the complement of the links is discussed. We prove that, when M is a homology sphere or when a link--in a generic manifold M--is homologically trivial, the associated observables coincide with the observables of the sphere S{sup 3}. Finally, we show that the U(1) Reshetikhin-Turaev surgery invariant of the manifold M is not a function of the homology group only, nor a function of the homotopy type of M alone.

  2. Fivebranes and 3-manifold homology

    DOE PAGES

    Gukov, Sergei; Putrov, Pavel; Vafa, Cumrun

    2017-07-14

    Motivated by physical constructions of homological knot invariants, we study their analogs for closed 3-manifolds. We show that vebrane compacti cations provide a universal description of various old and new homological invariants of 3-manifolds. In terms of 3d/3d correspondence, such invariants are given by the Q-cohomology of the Hilbert space of partially topologically twisted 3d N = 2 theory T[M3] on a Riemann surface with defects. We demonstrate this by concrete and explicit calculations in the case of monopole/Heegaard Floer homology and a 3-manifold analog of Khovanov-Rozansky link homology. The latter gives a categori cation of Chern-Simons partition function. Finally,more » some of the new key elements include the explicit form of the S-transform and a novel connection between categori cation and a previously mysterious role of Eichler integrals in Chern-Simons theory.« less

  3. The PIKE homolog Centaurin gamma regulates developmental timing in Drosophila.

    PubMed

    Gündner, Anna Lisa; Hahn, Ines; Sendscheid, Oliver; Aberle, Hermann; Hoch, Michael

    2014-01-01

    Phosphoinositide-3-kinase enhancer (PIKE) proteins encoded by the PIKE/CENTG1 gene are members of the gamma subgroup of the Centaurin superfamily of small GTPases. They are characterized by their chimeric protein domain architecture consisting of a pleckstrin homology (PH) domain, a GTPase-activating (GAP) domain, Ankyrin repeats as well as an intrinsic GTPase domain. In mammals, three PIKE isoforms with variations in protein structure and subcellular localization are encoded by the PIKE locus. PIKE inactivation in mice results in a broad range of defects, including neuronal cell death during brain development and misregulation of mammary gland development. PIKE -/- mutant mice are smaller, contain less white adipose tissue, and show insulin resistance due to misregulation of AMP-activated protein kinase (AMPK) and insulin receptor/Akt signaling. here, we have studied the role of PIKE proteins in metabolic regulation in the fly. We show that the Drosophila PIKE homolog, ceng1A, encodes functional GTPases whose internal GAP domains catalyze their GTPase activity. To elucidate the biological function of ceng1A in flies, we introduced a deletion in the ceng1A gene by homologous recombination that removes all predicted functional PIKE domains. We found that homozygous ceng1A mutant animals survive to adulthood. In contrast to PIKE -/- mouse mutants, genetic ablation of Drosophila ceng1A does not result in growth defects or weight reduction. Although metabolic pathways such as insulin signaling, sensitivity towards starvation and mobilization of lipids under high fed conditions are not perturbed in ceng1A mutants, homozygous ceng1A mutants show a prolonged development in second instar larval stage, leading to a late onset of pupariation. In line with these results we found that expression of ecdysone inducible genes is reduced in ceng1A mutants. Together, we propose a novel role for Drosophila Ceng1A in regulating ecdysone signaling-dependent second to third instar

  4. Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin.

    PubMed

    Chauvigné, François; Verdura, Sara; Mazón, María José; Boj, Mónica; Zanuy, Silvia; Gómez, Ana; Cerdà, Joan

    2015-09-15

    In flatfishes with asynchronous and semicystic spermatogenesis, such as the Senegalese sole (Solea senegalensis), the specific roles of the pituitary gonadotropins during germ cell development, particularly of the follicle-stimulating hormone (Fsh), are still largely unknown in part due to the lack of homologous immunoassays for this hormone. In this study, an enzyme-linked immunosorbent assay (ELISA) for Senegalese sole Fsh was developed by generating a rabbit antiserum against a recombinant chimeric single-chain Fsh molecule (rFsh-C) produced by the yeast Pichia pastoris. The rFsh-C N- and C-termini were formed by the mature sole Fsh β subunit (Fshβ) and the chicken glycoprotein hormone common α subunit (CGA), respectively. Depletion of the antiserum to remove anti-CGA antibodies further enriched the sole Fshβ-specific antibodies, which were used to develop the ELISA using the rFsh-C for the standard curve. The sensitivity of the assay was 10 and 50 pg/ml for Fsh measurement in plasma and pituitary, respectively, and the cross-reactivity with a homologous recombinant single-chain luteinizing hormone was 1%. The standard curve for rFsh-C paralleled those of serially diluted plasma and pituitary extracts of other flatfishes, such as the Atlantic halibut, common sole and turbot. In Senegalese sole males, the highest plasma Fsh levels were found during early spermatogenesis but declined during enhanced spermiation, as found in teleosts with cystic spermatogenesis. In pubertal males, however, the circulating Fsh levels were as high as in adult spermiating fish, but interestingly the Fsh receptor in the developing testis containing only spermatogonia was expressed in Leydig cells but not in the primordial Sertoli cells. These results indicate that a recombinant chimeric Fsh can be used to generate specific antibodies against the Fshβ subunit and to develop a highly sensitive ELISA for Fsh measurements in diverse flatfishes.

  5. Object-oriented Persistent Homology.

    PubMed

    Wang, Bao; Wei, Guo-Wei

    2016-01-15

    Persistent homology provides a new approach for the topological simplification of big data via measuring the life time of intrinsic topological features in a filtration process and has found its success in scientific and engineering applications. However, such a success is essentially limited to qualitative data classification and analysis. Indeed, persistent homology has rarely been employed for quantitative modeling and prediction. Additionally, the present persistent homology is a passive tool, rather than a proactive technique, for classification and analysis. In this work, we outline a general protocol to construct object-oriented persistent homology methods. By means of differential geometry theory of surfaces, we construct an objective functional, namely, a surface free energy defined on the data of interest. The minimization of the objective functional leads to a Laplace-Beltrami operator which generates a multiscale representation of the initial data and offers an objective oriented filtration process. The resulting differential geometry based object-oriented persistent homology is able to preserve desirable geometric features in the evolutionary filtration and enhances the corresponding topological persistence. The cubical complex based homology algorithm is employed in the present work to be compatible with the Cartesian representation of the Laplace-Beltrami flow. The proposed Laplace-Beltrami flow based persistent homology method is extensively validated. The consistence between Laplace-Beltrami flow based filtration and Euclidean distance based filtration is confirmed on the Vietoris-Rips complex for a large amount of numerical tests. The convergence and reliability of the present Laplace-Beltrami flow based cubical complex filtration approach are analyzed over various spatial and temporal mesh sizes. The Laplace-Beltrami flow based persistent homology approach is utilized to study the intrinsic topology of proteins and fullerene molecules. Based on a

  6. Object-oriented Persistent Homology

    PubMed Central

    Wang, Bao; Wei, Guo-Wei

    2015-01-01

    Persistent homology provides a new approach for the topological simplification of big data via measuring the life time of intrinsic topological features in a filtration process and has found its success in scientific and engineering applications. However, such a success is essentially limited to qualitative data classification and analysis. Indeed, persistent homology has rarely been employed for quantitative modeling and prediction. Additionally, the present persistent homology is a passive tool, rather than a proactive technique, for classification and analysis. In this work, we outline a general protocol to construct object-oriented persistent homology methods. By means of differential geometry theory of surfaces, we construct an objective functional, namely, a surface free energy defined on the data of interest. The minimization of the objective functional leads to a Laplace-Beltrami operator which generates a multiscale representation of the initial data and offers an objective oriented filtration process. The resulting differential geometry based object-oriented persistent homology is able to preserve desirable geometric features in the evolutionary filtration and enhances the corresponding topological persistence. The cubical complex based homology algorithm is employed in the present work to be compatible with the Cartesian representation of the Laplace-Beltrami flow. The proposed Laplace-Beltrami flow based persistent homology method is extensively validated. The consistence between Laplace-Beltrami flow based filtration and Euclidean distance based filtration is confirmed on the Vietoris-Rips complex for a large amount of numerical tests. The convergence and reliability of the present Laplace-Beltrami flow based cubical complex filtration approach are analyzed over various spatial and temporal mesh sizes. The Laplace-Beltrami flow based persistent homology approach is utilized to study the intrinsic topology of proteins and fullerene molecules. Based on a

  7. Object-oriented persistent homology

    NASA Astrophysics Data System (ADS)

    Wang, Bao; Wei, Guo-Wei

    2016-01-01

    Persistent homology provides a new approach for the topological simplification of big data via measuring the life time of intrinsic topological features in a filtration process and has found its success in scientific and engineering applications. However, such a success is essentially limited to qualitative data classification and analysis. Indeed, persistent homology has rarely been employed for quantitative modeling and prediction. Additionally, the present persistent homology is a passive tool, rather than a proactive technique, for classification and analysis. In this work, we outline a general protocol to construct object-oriented persistent homology methods. By means of differential geometry theory of surfaces, we construct an objective functional, namely, a surface free energy defined on the data of interest. The minimization of the objective functional leads to a Laplace-Beltrami operator which generates a multiscale representation of the initial data and offers an objective oriented filtration process. The resulting differential geometry based object-oriented persistent homology is able to preserve desirable geometric features in the evolutionary filtration and enhances the corresponding topological persistence. The cubical complex based homology algorithm is employed in the present work to be compatible with the Cartesian representation of the Laplace-Beltrami flow. The proposed Laplace-Beltrami flow based persistent homology method is extensively validated. The consistence between Laplace-Beltrami flow based filtration and Euclidean distance based filtration is confirmed on the Vietoris-Rips complex for a large amount of numerical tests. The convergence and reliability of the present Laplace-Beltrami flow based cubical complex filtration approach are analyzed over various spatial and temporal mesh sizes. The Laplace-Beltrami flow based persistent homology approach is utilized to study the intrinsic topology of proteins and fullerene molecules. Based on a

  8. Co-expression of multiple target proteins in plants from a tobacco mosaic virus vector using a combination of homologous and heterologous subgenomic promoters.

    PubMed

    Roy, Gourgopal; Weisburg, Sangeetha; Foy, Kelly; Rabindran, Shailaja; Mett, Vadim; Yusibov, Vidadi

    2011-11-01

    To co-express multiple target proteins, we engineered a single-component chimeric tobacco mosaic virus (TMV)-based vector containing homologous and heterologous capsid protein subgenomic RNA promoters. Delivery of this vector into Nicotiana benthamiana plants via agroinfiltration resulted in co-expression of two reporter genes within a single cell. Furthermore, co-expression of a host-specific antisense RNA or a silencing suppressor protein from this vector augmented the accumulation of green fluorescent protein or a vaccine antigen, hemagglutinin from avian influenza virus A/Vietnam/1194/04. These findings suggest that this chimeric vector utilizing the homologous and heterologous subgenomic TMV promoters has a potential for high-level production of multiple therapeutic proteins including monoclonal antibodies.

  9. The semaphorontic view of homology

    PubMed Central

    Assis, Leandro C.S.; Rieppel, Olivier

    2015-01-01

    ABSTRACT The relation of homology is generally characterized as an identity relation, or alternatively as a correspondence relation, both of which are transitive. We use the example of the ontogenetic development and evolutionary origin of the gnathostome jaw to discuss identity and transitivity of the homology relation under the transformationist and emergentist paradigms respectively. Token identity and consequent transitivity of homology relations are shown to be requirements that are too strong to allow the origin of genuine evolutionary novelties. We consequently introduce the concept of compositional identity that is grounded in relations prevailing between parts (organs and organ systems) of a whole (organism). We recognize an ontogenetic identity of parts within a whole throughout the sequence of successive developmental stages of those parts: this is an intra‐organismal character identity maintained throughout developmental trajectory. Correspondingly, we recognize a phylogenetic identity of homologous parts within two or more organisms of different species: this is an inter‐species character identity maintained throughout evolutionary trajectory. These different dimensions of character identity—ontogenetic (through development) and phylogenetic (via shared evolutionary history)—break the transitivity of homology relations. Under the transformationist paradigm, the relation of homology reigns over the entire character (‐state) transformation series, and thus encompasses the plesiomorphic as well as the apomorphic condition of form. In contrast, genuine evolutionary novelties originate not through transformation of ancestral characters (‐states), but instead through deviating developmental trajectories that result in alternate characters. Under the emergentist paradigm, homology is thus synonymous with synapomorphy. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 578–587, 2015. © 2015 The Authors. Journal of Experimental Zoology Part B: Molecular and

  10. The semaphorontic view of homology.

    PubMed

    Havstad, Joyce C; Assis, Leandro C S; Rieppel, Olivier

    2015-11-01

    The relation of homology is generally characterized as an identity relation, or alternatively as a correspondence relation, both of which are transitive. We use the example of the ontogenetic development and evolutionary origin of the gnathostome jaw to discuss identity and transitivity of the homology relation under the transformationist and emergentist paradigms respectively. Token identity and consequent transitivity of homology relations are shown to be requirements that are too strong to allow the origin of genuine evolutionary novelties. We consequently introduce the concept of compositional identity that is grounded in relations prevailing between parts (organs and organ systems) of a whole (organism). We recognize an ontogenetic identity of parts within a whole throughout the sequence of successive developmental stages of those parts: this is an intra-organismal character identity maintained throughout developmental trajectory. Correspondingly, we recognize a phylogenetic identity of homologous parts within two or more organisms of different species: this is an inter-species character identity maintained throughout evolutionary trajectory. These different dimensions of character identity--ontogenetic (through development) and phylogenetic (via shared evolutionary history)--break the transitivity of homology relations. Under the transformationist paradigm, the relation of homology reigns over the entire character (-state) transformation series, and thus encompasses the plesiomorphic as well as the apomorphic condition of form. In contrast, genuine evolutionary novelties originate not through transformation of ancestral characters (-states), but instead through deviating developmental trajectories that result in alternate characters. Under the emergentist paradigm, homology is thus synonymous with synapomorphy. © 2015 The Authors. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution Published by Wiley Periodicals, Inc.

  11. Novel Antiproliferative Chimeric Compounds with Marked Histone Deacetylase Inhibitory Activity

    PubMed Central

    2014-01-01

    Given our interest in finding potential antitumor agents and in view of the multifactorial mechanistic nature of cancer, in the present work, taking advantage of the multifunctional ligands approach, new chimeric molecules were designed and synthesized by combining in single chemical entities structural features of SAHA, targeting histone deacetylases (HDACs), with substituted stilbene or terphenyl derivatives previously obtained by us and endowed with antiproliferative and pro-apoptotic activity. The new chimeric derivatives were characterized with respect to their cytotoxic activity and their effects on cell cycle progression on different tumor cell lines, as well as their HDACs inhibition. Among the other, trans-6 showed the most interesting biological profile, as it exhibited a strong pro-apoptotic activity in tumor cell lines in comparison with both of its parent compounds and a marked HDAC inhibition. PMID:25221651

  12. Chimeric antigen receptors: driving immunology towards synthetic biology

    PubMed Central

    Sadelain, Michel

    2017-01-01

    The advent of second generation CARs and the CD19 paradigm have ushered a new therapeutic modality in oncology. In contrast to earlier forms of adoptive cell therapy, which were based on the isolation and expansion of naturally occurring T cells, CAR therapy is based on the design and manufacture of engineered T cells with optimized properties. A new armamentarium, comprising not only CARs but also chimeric costimulatory receptors, chimeric cytokine receptors, inhibitory receptors and synthetic Notch receptors, expressed in naïve, central memory or stem cell-like memory T cells, is being developed for clinical use in a wide range of cancers. Immunological principles are thus finding a new purpose thanks to advances in genetic engineering, synthetic biology and cell manufacturing sciences. PMID:27372731

  13. Birth of two chimeric genes in the Hominidae lineage.

    PubMed

    Courseaux, A; Nahon, J L

    2001-02-16

    How genes with newly characterized functions originate remains a fundamental question. PMCHL1 and PMCHL2, two chimeric genes derived from the melanin-concentrating hormone (MCH) gene, offer an opportunity to examine such an issue in the human lineage. Detailed structural, expression, and phylogenetic analysis showed that the PMCHL1 gene was created near 25 million years ago (Ma) by a complex mechanism of exon shuffling through retrotransposition of an antisense MCH messenger RNA coupled to de novo creation of splice sites. PMCHL2 arose 5 to 10 Ma by an event of duplication involving a large chromosomal region encompassing the PMCHL1 locus. The RNA expression patterns of those chimeric genes suggest that they have been submitted to strong regulatory constraints during primate evolution.

  14. Blood Chimerism in Dizygotic Monochorionic Twins During 5 Years Observation.

    PubMed

    Dziegiel, M H; Hansen, M H; Haedersdal, S; Barrett, A N; Rieneck, K; Main, K M; Hansen, A T; Clausen, F B

    2017-04-19

    Dizygotic monochorionic twin pregnancies can result in blood chimerism due to in utero twin-to-twin exchange of stem cells. In this case, we examined the proportion of allogeneic red blood cells by flow cytometry and the proportion of allogeneic nucleated cells by digital polymerase chain reaction at 7 months and again at 5 years. We found an increase in the proportion of allogeneic cells from 63% to 89% in one twin, and a similar increase in autologous cells in the other twin from 57% to 84%. A paradigm for stem cell therapy could be modeled on this case: induction of tolerance and chimerism by antenatal transfusion of donor stem cells. The procedure would hold the promise of transplantation and tolerance induction without myeloablative conditioning for inheritable benign hematological diseases such as sickle cell disease and thalassemia. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

  15. Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis.

    PubMed

    Gryczan, T; Shivakumar, A G; Dubnau, D

    1980-01-01

    Restriction endonuclease cleavage maps of seven chimeric plasmids that may be used for molecular cloning in Bacillus subtilis are presented. These plasmids all carry multiple antibiotic resistance markers and were constructed by in vitro molecular cloning techniques. Several of the antibiotic resistance markers were shown to undergo insertional inactivation at specific restriction endonuclease sites. Kanamycin inactivation occurred at the BglII site of pUB110 derivatives, erythromycin inactivation occurred at the HpaI and BclI sites of pE194 derivatives, and streptomycin inactivation occurred at the HindIII site of pSA0501 derivatives. A stable mini-derivative of pBD12 was isolated and characterized. By using these plasmids, we identified proteins involved in plasmid-coded kanamycin and erythromycin resistance. The properties and uses of these chimeric plasmids in the further development of recombinant deoxyribonucleic acid technology in B. subtilis are discussed.

  16. Mechanisms of Tolerance Induction by Hematopoietic Chimerism: The Immune Perspective.

    PubMed

    Yolcu, Esma S; Shirwan, Haval; Askenasy, Nadir

    2017-03-01

    Hematopoietic chimerism is one of the effective approaches to induce tolerance to donor-derived tissue and organ grafts without administration of life-long immunosuppressive therapy. Although experimental efforts to develop such regimens have been ongoing for decades, substantial cumulative toxicity of combined hematopoietic and tissue transplants precludes wide clinical implementation. Tolerance is an active immunological process that includes both peripheral and central mechanisms of mutual education of coresident donor and host immune systems. The major stages include sequential suppression of early alloreactivity, establishment of hematopoietic chimerism and suppressor cells that sustain the state of tolerance, with significant mechanistic and temporal overlap along the tolerization process. Efforts to devise less toxic transplant strategies by reduction of preparatory conditioning focus on modulation rather than deletion of residual host immunity and early reinstitution of regulatory subsets at the central and peripheral levels. Stem Cells Translational Medicine 2017;6:700-712.

  17. Genetic engineering of chimeric antigen receptors using lamprey derived variable lymphocyte receptors

    PubMed Central

    Moot, Robert; Raikar, Sunil S; Fleischer, Lauren; Querrey, Melissa; Tylawsky, Daniel E; Nakahara, Hirotomo; Doering, Christopher B; Spencer, H Trent

    2016-01-01

    Chimeric antigen receptors (CARs) are used to redirect effector cell specificity to selected cell surface antigens. Using CARs, antitumor activity can be initiated in patients with no prior tumor specific immunity. Although CARs have shown promising clinical results, the technology remains limited by the availability of specific cognate cell target antigens. To increase the repertoire of targetable tumor cell antigens we utilized the immune system of the sea lamprey to generate directed variable lymphocyte receptors (VLRs). VLRs serve as membrane bound and soluble immune effectors analogous but not homologous to immunoglobulins. They have a fundamentally different structure than immunoglobulin (Ig)-based antibodies while still demonstrating high degrees of specificity and affinity. To test the functionality of VLRs as the antigen recognition domain of CARs, two VLR-CARs were created. One contained a VLR specific for a murine B cell leukemia and the other contained a VLR specific for the human T cell surface antigen, CD5. The CAR design consisted of the VLR sequence, myc-epitope tag, CD28 transmembrane domain, and intracellular CD3ζ signaling domain. We demonstrate proof of concept, including gene transfer, biosynthesis, cell surface localization, and effector cell activation for multiple VLR-CAR designs. Therefore, VLRs provide an alternative means of CAR-based cancer recognition. PMID:27933313

  18. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    NASA Astrophysics Data System (ADS)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  19. Adaptive impact of the chimeric gene Quetzalcoatl in Drosophila melanogaster

    PubMed Central

    Rogers, Rebekah L.; Bedford, Trevor; Lyons, Ana M.; Hartl, Daniel L.

    2010-01-01

    Chimeric genes, which form through the genomic fusion of two protein-coding genes, are a significant source of evolutionary novelty in Drosophila melanogaster. However, the propensity of chimeric genes to produce adaptive phenotypic changes is not fully understood. Here, we describe the chimeric gene Quetzalcoatl (Qtzl; CG31864), which formed in the recent past and swept to fixation in D. melanogaster. Qtzl arose through a duplication on chromosome 2L that united a portion of the mitochondrially targeted peptide CG12264 with a segment of the polycomb gene escl. The 3′ segment of the gene, which is derived from escl, is inherited out of frame, producing a unique peptide sequence. Nucleotide diversity is drastically reduced and site frequency spectra are significantly skewed surrounding the duplicated region, a finding consistent with a selective sweep on the duplicate region containing Qtzl. Qtzl has an expression profile that largely resembles that of escl, with expression in early pupae, adult females, and male testes. However, expression patterns appear to have been decoupled from both parental genes during later embryonic development and in head tissues of adult males, indicating that Qtzl has developed a distinct regulatory profile through the rearrangement of different 5′ and 3′ regulatory domains. Furthermore, misexpression of Qtzl suppresses defects in the formation of the neuromuscular junction in larvae, demonstrating that Qtzl can produce phenotypic effects in cells. Together, these results show that chimeric genes can produce structural and regulatory changes in a single mutational step and may be a major factor in adaptive evolution. PMID:20534482

  20. Cord blood chimerism and relapse after haplo-cord transplantation.

    PubMed

    van Besien, Koen; Koshy, Nebu; Gergis, Usama; Mayer, Sebastian; Cushing, Melissa; Rennert, Hannah; Reich-Slotky, Ronit; Mark, Tomer; Pearse, Roger; Rossi, Adriana; Phillips, Adrienne; Vasovic, Liljana; Ferrante, Rosanna; Hsu, Yen-Michael; Shore, Tsiporah

    2017-02-01

    Haplo-cord stem cell transplantation combines the infusion of CD34 selected hematopoietic progenitors from a haplo-identical donor with an umbilical cord blood (UCB) graft from an unrelated donor and allows faster count recovery, with low rates of disease recurrence and chronic graft-versus-host disease (GVHD). But the contribution of the umbilical cord blood graft to long-term transplant outcome remains unclear. We analyzed 39 recipients of haplo-cord transplants with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), engrafted and in remission at 2 months. Median age was 66 (18-72) and all had intermediate, high, or very-high risk disease. Less than 20% UCB chimerism in the CD33 lineage was associated with an increased rate of disease recurrence (54% versus 11% p < 0.0001) and decrease in one year progression-free (20% versus 55%, p = 0.004) and overall survival (30% versus 62%, p = 0.02). Less than 100% UCB chimerism in the CD3 lineage was associated with increase rate of disease recurrence (46% versus 12%, p = 0.007). Persistent haplo-chimerism in the CD3 lineage was associated with an increased rate of disease recurrence (40% versus 15%, p = 0.009) Chimerism did not predict for treatment related mortality. The cumulative incidence of acute GVHD by day 100 was 43%. The cumulative incidence of moderate/severe chronic GVHD was only 5%. Engraftment of the umbilical cord blood grafts provides powerful graft-versus-leukemia (GVL) effects which protect against disease recurrence and is associated with low risk of chronic GVHD. Engraftment of CD34 selected haplo-identical cells can lead to rapid development of circulating T-cells, but when these cells dominate, GVL-effects are limited and rates of disease recurrence are high.

  1. Immunogenicity of candidate chimeric DNA vaccine against tuberculosis and leishmaniasis.

    PubMed

    Dey, Ayan; Kumar, Umesh; Sharma, Pawan; Singh, Sarman

    2009-08-13

    Mycobacterium tuberculosis and Leishmania donovani are important intracellular pathogens, especially in Indian context. In India and other South East Asian countries, both these infections are highly endemic and in about 20% cases co-infection of these pathogens is reported. For both these pathogens cell mediated immunity plays most important role. The available treatment of these infections is either prolonged or cumbersome or it is ineffective in controlling the outbreaks and spread. Therefore, potentiation of a common host defense mechanism can be used to prevent both the infections simultaneously. In this study we have developed a novel chimeric DNA vaccine candidate comprising the esat-6 gene of M. tuberculosis and kinesin motor domain gene of L. donovani. After developing this novel chimera, its immunogenicity was studied in mouse model. The immune response was compared with individual constructs of esat-6 and kinesin motor domain. The results showed that immunization with chimeric DNA vaccine construct resulted in stronger IFN-gamma and IL-2 response against kinesin (3012+/-102 and 367.5+/-8.92pg/ml) and ESAT-6 (1334+/-46.5 and 245.1+/-7.72pg/ml) in comparison to the individual vaccine constructs. The reciprocal immune response (IFN-gamma and IL-2) against individual construct was lower (kinesin motor domain: 1788+/-36.48 and 341.8+/-9.801pg/ml and ESAT-6: 867.0+/-47.23 and 170.8+/-4.578pg/ml, respectively). The results also suggest that using the chimeric construct both proteins yielded a reciprocal adjuvant affect over each other as the IFN-gamma production against chimera vaccination is statistically significant (p<0.0001) than individual construct vaccination. From this pilot study we could envisage that the chimeric DNA vaccine construct may offer an attractive strategy in controlling co-infection of leishmaniasis and tuberculosis and have important implication in future vaccine design.

  2. Chimeric Protein Complexes in Hybrid Species Generate Novel Phenotypes

    PubMed Central

    Piatkowska, Elzbieta M.; Naseeb, Samina; Knight, David; Delneri, Daniela

    2013-01-01

    Hybridization between species is an important mechanism for the origin of novel lineages and adaptation to new environments. Increased allelic variation and modification of the transcriptional network are the two recognized forces currently deemed to be responsible for the phenotypic properties seen in hybrids. However, since the majority of the biological functions in a cell are carried out by protein complexes, inter-specific protein assemblies therefore represent another important source of natural variation upon which evolutionary forces can act. Here we studied the composition of six protein complexes in two different Saccharomyces “sensu stricto” hybrids, to understand whether chimeric interactions can be freely formed in the cell in spite of species-specific co-evolutionary forces, and whether the different types of complexes cause a change in hybrid fitness. The protein assemblies were isolated from the hybrids via affinity chromatography and identified via mass spectrometry. We found evidence of spontaneous chimericity for four of the six protein assemblies tested and we showed that different types of complexes can cause a variety of phenotypes in selected environments. In the case of TRP2/TRP3 complex, the effect of such chimeric formation resulted in the fitness advantage of the hybrid in an environment lacking tryptophan, while only one type of parental combination of the MBF complex allowed the hybrid to grow under respiratory conditions. These phenotypes were dependent on both genetic and environmental backgrounds. This study provides empirical evidence that chimeric protein complexes can freely assemble in cells and reveals a new mechanism to generate phenotypic novelty and plasticity in hybrids to complement the genomic innovation resulting from gene duplication. The ability to exchange orthologous members has also important implications for the adaptation and subsequent genome evolution of the hybrids in terms of pattern of gene loss. PMID

  3. Chimeric plantibody passively protects mice against aerosolized ricin challenge.

    PubMed

    Sully, Erin K; Whaley, Kevin J; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do H; Pauly, Michael H; Velasco, Jesus; Hiatt, Ernie; Morton, Josh; Swope, Kelsi; Roy, Chad J; Zeitlin, Larry; Mantis, Nicholas J

    2014-05-01

    Recent incidents in the United States and abroad have heightened concerns about the use of ricin toxin as a bioterrorism agent. In this study, we produced, using a robust plant-based platform, four chimeric toxin-neutralizing monoclonal antibodies that were then evaluated for the ability to passively protect mice from a lethal-dose ricin challenge. The most effective antibody, c-PB10, was further evaluated in mice as a therapeutic following ricin exposure by injection and inhalation.

  4. Prism adaptation changes perceptual awareness for chimeric visual objects but not for chimeric faces in spatial neglect after right-hemisphere stroke.

    PubMed

    Sarri, Margarita; Kalra, Lalit; Greenwood, Richard; Driver, Jon

    2006-06-01

    Prism adaptation can ameliorate some symptoms of left spatial neglect after right-hemisphere stroke. The mechanisms behind this remain unclear. Prism therapy may increase exploration towards the contralesional side, yet without improving perceptual awareness, as apparently for the left side of chimeric face stimuli (Ferber et al. 2003). However, other prism studies suggest that perceptual awareness might be improved (e.g., Maravita et al., 2003). We tested the impact of prism therapy on visual awareness for the left side of chimeric objects as well as chimeric faces, in three neglect patients. Prism therapy dramatically improved awareness for the identity of the left side of chimeric non-face objects, but had no effect on judging expressions for chimeric faces. The latter may thus be unique in showing no prism benefit.

  5. Xenobiotics, chimerism and the induction of tolerance following organ transplantation

    PubMed Central

    Delaney, C. P.; Thomson, A. W.; Demetris, A. J.; Starzl, T. E.

    2010-01-01

    Summary The successful results seen after organ transplantation are largely attributable to the potency and specificity of modern immunosuppressive agents. Although drug-free unresponsiveness to graft alloantigens has not been routinely achieved in clinical practice, recent appreciation of the importance of cell chimerism, which develops after the migration from donor to host of leukocytes contained in solid organ grafts, has introduced a concept which may explain the mechanism of graft tolerance. Recent evidence has indicated that immunosuppressive drugs may have a common potential to induce graft tolerance, even though they act through diverse mechanisms, and that this potential may be mediated by a permissive effect on the migration and survival of donor-derived leukocytes. This review briefly examines the mechanisms by which immunosuppressive drugs function and analyses the different methods which these agents might use to induce chimerism associated with graft tolerance. Furthermore, we describe ongoing clinical studies in which the chimerism produced after solid organ transplantation is augmented with donor bone marrow in an attempt to facilitate the induction of tolerance. PMID:7584491

  6. Chimeric antibodies with extended half-life in ferrets

    PubMed Central

    Nesspor, Thomas C; Scallon, Bernard

    2014-01-01

    Background Ferrets have long been used as a disease model for the study of influenza vaccines, but a more recent use has been for the study of human monoclonal antibodies directed against influenza viruses. Published data suggest that human antibodies are cleared unusually quickly from the ferret and that immune responses may be partially responsible. This immunogenicity increases variability within groups and may present an obstacle to long-term studies. Objective Our aim was to identify an antibody design with reduced immunogenicity and longer circulating half-life in ferrets. Methods The constant region coding sequences for ferret immunoglobulin G were cloned, and chimeric human/ferret antibodies were expressed and purified. Some of the chimeric antibodies included substitutions that have been shown to extend the half-life of human IgG antibodies. These chimeric antibodies were tested for binding to recombinant ferret FcRn receptor and then evaluated in pharmacokinetic studies in ferrets. Results A one-residue substitution in the ferret Fc domain, S252Y, was identified that increased binding affinity to the ferret neonatal receptor by 24-fold and extended half-life from 65 ± 27 to 206 ± 28 hours or ∼9 days. Ferrets dosed twice with this surrogate antibody showed no indications of an immune response. Conclusion Expressing the variable region of a candidate human therapeutic antibody with ferret constant regions containing the S252Y substitution can offer long half-life and limit immunogenicity. PMID:25074755

  7. Chimeric creatures in Greek mythology and reflections in science.

    PubMed

    Bazopoulou-Kyrkanidou, E

    2001-04-15

    "The Chimaera" in Homer's Iliad, "was of divine stock, not of men, in the forepart a lion, in the hinder a serpent, and in the midst a goat, ellipsis Bellerophon slew her, trusting in the signs of the gods." In Hesiod's Theogony it is emphasized that "Chimaera ellipsis had three heads, one of a grim-eyed lion, another of a goat, and another of a snakeellipsis". In addition to this interspecies animal chimera, human/animal chimeras are referred to in Greek mythology, preeminent among them the Centaurs and the Minotaur. The Centaurs, as horse/men, first appear in Geometric and early Archaic art, but in the literature not until early in the fifth century B.C. The bullheaded-man Minotaur, who is not certainly attested in the literary evidence until circa 500 B.C., first appears in art about 650 B.C. Attempts, in the fourth century B.C. and thereafter, to rationalize their mythical appearance were in vain; their chimeric nature retained its fascinating and archetypal form over the centuries. Early in the 1980s, experimental sheep/goat chimeras were produced removing the reproductive barrier between these two animal species. Late in the 1990s, legal, political, ethical, and moral fights loomed over a patent bid on human/animal chimeras. Chimeric technology is recently developed; however, the concept of chimerism has existed in literary and artistic form in ancient mythology. This is yet another example where art and literature precede scientific research and development.

  8. A modular strategy for engineering orthogonal chimeric RNA transcription regulators

    PubMed Central

    Takahashi, Melissa K.; Lucks, Julius B.

    2013-01-01

    Antisense RNA transcription attenuators are a key component of the synthetic biology toolbox, with their ability to serve as building blocks for both signal integration logic circuits and transcriptional cascades. However, a central challenge to building more sophisticated RNA genetic circuitry is creating larger families of orthogonal attenuators that function independently of each other. Here, we overcome this challenge by developing a modular strategy to create chimeric fusions between the engineered transcriptional attenuator from plasmid pT181 and natural antisense RNA translational regulators. Using in vivo gene expression assays in Escherichia coli, we demonstrate our ability to create chimeric attenuators by fusing sequences from five different translational regulators. Mutagenesis of these functional attenuators allowed us to create a total of 11 new chimeric attenutaors. A comprehensive orthogonality test of these culminated in a 7 × 7 matrix of mutually orthogonal regulators. A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation. We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry. PMID:23761434

  9. Genomic homologous recombination in planta.

    PubMed Central

    Gal, S; Pisan, B; Hohn, T; Grimsley, N; Hohn, B

    1991-01-01

    A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process. Images PMID:2026150

  10. Lassa-vesicular stomatitis chimeric virus safely destroys brain tumors.

    PubMed

    Wollmann, Guido; Drokhlyansky, Eugene; Davis, John N; Cepko, Connie; van den Pol, Anthony N

    2015-07-01

    High-grade tumors in the brain are among the deadliest of cancers. Here, we took a promising oncolytic virus, vesicular stomatitis virus (VSV), and tested the hypothesis that the neurotoxicity associated with the virus could be eliminated without blocking its oncolytic potential in the brain by replacing the neurotropic VSV glycoprotein with the glycoprotein from one of five different viruses, including Ebola virus, Marburg virus, lymphocytic choriomeningitis virus (LCMV), rabies virus, and Lassa virus. Based on in vitro infections of normal and tumor cells, we selected two viruses to test in vivo. Wild-type VSV was lethal when injected directly into the brain. In contrast, a novel chimeric virus (VSV-LASV-GPC) containing genes from both the Lassa virus glycoprotein precursor (GPC) and VSV showed no adverse actions within or outside the brain and targeted and completely destroyed brain cancer, including high-grade glioblastoma and melanoma, even in metastatic cancer models. When mice had two brain tumors, intratumoral VSV-LASV-GPC injection in one tumor (glioma or melanoma) led to complete tumor destruction; importantly, the virus moved contralaterally within the brain to selectively infect the second noninjected tumor. A chimeric virus combining VSV genes with the gene coding for the Ebola virus glycoprotein was safe in the brain and also selectively targeted brain tumors but was substantially less effective in destroying brain tumors and prolonging survival of tumor-bearing mice. A tropism for multiple cancer types combined with an exquisite tumor specificity opens a new door to widespread application of VSV-LASV-GPC as a safe and efficacious oncolytic chimeric virus within the brain. Many viruses have been tested for their ability to target and kill cancer cells. Vesicular stomatitis virus (VSV) has shown substantial promise, but a key problem is that if it enters the brain, it can generate adverse neurologic consequences, including death. We tested a series of

  11. Lassa-Vesicular Stomatitis Chimeric Virus Safely Destroys Brain Tumors

    PubMed Central

    Wollmann, Guido; Drokhlyansky, Eugene; Davis, John N.; Cepko, Connie

    2015-01-01

    ABSTRACT High-grade tumors in the brain are among the deadliest of cancers. Here, we took a promising oncolytic virus, vesicular stomatitis virus (VSV), and tested the hypothesis that the neurotoxicity associated with the virus could be eliminated without blocking its oncolytic potential in the brain by replacing the neurotropic VSV glycoprotein with the glycoprotein from one of five different viruses, including Ebola virus, Marburg virus, lymphocytic choriomeningitis virus (LCMV), rabies virus, and Lassa virus. Based on in vitro infections of normal and tumor cells, we selected two viruses to test in vivo. Wild-type VSV was lethal when injected directly into the brain. In contrast, a novel chimeric virus (VSV-LASV-GPC) containing genes from both the Lassa virus glycoprotein precursor (GPC) and VSV showed no adverse actions within or outside the brain and targeted and completely destroyed brain cancer, including high-grade glioblastoma and melanoma, even in metastatic cancer models. When mice had two brain tumors, intratumoral VSV-LASV-GPC injection in one tumor (glioma or melanoma) led to complete tumor destruction; importantly, the virus moved contralaterally within the brain to selectively infect the second noninjected tumor. A chimeric virus combining VSV genes with the gene coding for the Ebola virus glycoprotein was safe in the brain and also selectively targeted brain tumors but was substantially less effective in destroying brain tumors and prolonging survival of tumor-bearing mice. A tropism for multiple cancer types combined with an exquisite tumor specificity opens a new door to widespread application of VSV-LASV-GPC as a safe and efficacious oncolytic chimeric virus within the brain. IMPORTANCE Many viruses have been tested for their ability to target and kill cancer cells. Vesicular stomatitis virus (VSV) has shown substantial promise, but a key problem is that if it enters the brain, it can generate adverse neurologic consequences, including death. We

  12. Utilizing chimeric proteins for exploring the cellular fate of endogenous proteins.

    PubMed

    Ben-Yehudah, Ahmi; Aqeilan, Rami; Belostotsky, Ruth; Azar, Yehudith; Lorberboum-Galski, Haya

    2002-01-11

    We recently designed and constructed chimeric proteins for the elimination of specific cell populations. These chimeric proteins are composed of a targeting component fused to an apoptotic protein as the killing moiety. However, chimeric proteins can serve not only to eliminate cell populations, but also as "biological tools" for studying the fate of endogenous proteins. We show here that upon entering their target cell, a variety of chimeric proteins composed of an endogenous protein as their killing moiety reach the subcellular location of their endogenous counterpart. In contrast, bacterial-based killing domains head for the subcellular site of their substrate. Moreover, the chimeric protein acts similarly to the endogenous protein, while causing the cell to die. Therefore, chimeric proteins may serve as a unique tool for investigating cellular proteins and their intracellular localization, without the need to overexpress them.

  13. Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs.

    PubMed

    Zhao, Bingxin; Pan, Xiaoxia; Teng, Yumei; Xia, Wenyue; Wang, Jing; Wen, Yuling; Chen, Yuanding

    2015-10-01

    VP7 of group A rotavirus (RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector, three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains. Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.

  14. Chimeric Proteins to Detect DNA Damage and Mismatches

    SciTech Connect

    McCutchen-Maloney, S; Malfatti, M; Robbins, K M

    2002-01-14

    The goal of this project was to develop chimeric proteins composed of a DNA mismatch or damage binding protein and a nuclease, as well as methods to detect DNA mismatches and damage. We accomplished this through protein engineering based on using polymerase chain reactions (PCRs) to create chimeras with novel functions for damage and mismatch detection. This project addressed fundamental questions relating to disease susceptibility and radiation-induced damage in cells. It also supported and enhanced LLNL's competency in the emerging field of proteomics. In nature, DNA is constantly being subjected to damaging agents such as exposure to ultraviolet (UV) radiation and various environmental and dietary carcinogens. If DNA damage is not repaired however, mutations in DNA result that can eventually manifest in cancer and other diseases. In addition to damage-induced DNA mutations, single nucleotide polymorphisms (SNPs), which are variations in the genetic sequence between individuals, may predispose some to disease. As a result of the Human Genome Project, the integrity of a person's DNA can now be monitored. Therefore, methods to detect DNA damage, mutations, and SNPs are useful not only in basic research but also in the health and biotechnology industries. Current methods of detection often use radioactive labeling and rely on expensive instrumentation that is not readily available in many research settings. Our methods to detect DNA damage and mismatches employ simple gel electrophoresis and flow cytometry, thereby alleviating the need for radioactive labeling and expensive equipment. In FY2001, we explored SNP detection by developing methods based on the ability of the chimeric proteins to detect mismatches. Using multiplex assays with flow cytometry and fluorescent beads to which the DNA substrates where attached, we showed that several of the chimeras possess greater affinity for damaged and mismatched DNA than for native DNA. This affinity was demonstrated in

  15. Expression and purification of toxic anti-breast cancer p28-NRC chimeric protein

    PubMed Central

    Soleimani, Meysam; Mirmohammad-Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Jahanian-Najafabadi, Ali

    2016-01-01

    Background: Chimeric proteins consisting of a targeting moiety and a cytotoxic moiety are now under intense research focus for targeted therapy of cancer. Here, we report cloning, expression, and purification of such a targeted chimeric protein made up of p28 peptide as both targeting and anticancer moiety fused to NRC peptide as a cytotoxic moiety. However, since the antimicrobial activity of the NRC peptide would intervene expression of the chimeric protein in Escherichia coli, we evaluated the effects of two fusion tags, that is, thioredoxin (Trx) and 6x-His tags, and various expression conditions, on the expression of p28-NRC chimeric protein. Materials and Methods: In order to express the chimeric protein with only 6x-His tag, pET28 expression plasmid was used. Cloning in pET32 expression plasmid was performed to add both Trx and 6x-His tags to the chimeric protein. Expression of the chimeric protein with both plasmids was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis following optimization of expression conditions and host strains. Results: Expression of the chimeric protein in pET28a was performed. However, expression yield of the chimeric protein was low. Optimization of culture conditions and host strains led to reasonable expression yield of the toxic chimeric protein in pET32a vector. In cases of both plasmids, approximately 10 kDa deviation of the apparent molecular weight from the theoretical one was seen in SDS-PAGE of purified chimeric proteins. Conclusions: The study leads to proper expression and purification yield of p28-NRC chimeric protein with Trx tag following optimizing culture conditions and host strains. PMID:27169101

  16. Establishment of Donor Chimerism Using Allogeneic Bone Marrow with AMP Cell Co-infusion

    DTIC Science & Technology

    2016-09-01

    AWARD NUMBER: W81XWH-15-1-0234 TITLE: “Establishment of donor Chimerism Using Allogeneic Bone Marrow with AMP Cell Co-infusion” PRINCIPAL...Form 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18 Megan.sykes@columbia.edu Establishment of donor Chimerism Using Allogeneic Bone Marrow with AMP...promote the induction of durable mixed allogeneic chimerism in non-human primates. To this end, we have performed bone marrow transplantation in two

  17. Germ-line chimerism and paternal care in marmosets (Callithrix kuhlii).

    PubMed

    Ross, C N; French, J A; Ortí, G

    2007-04-10

    The formation of viable genetic chimeras in mammals through the transfer of cells between siblings in utero is rare. Using microsatellite DNA markers, we show here that chimerism in marmoset (Callithrix kuhlii) twins is not limited to blood-derived hematopoietic tissues as was previously described. All somatic tissue types sampled were found to be chimeric. Notably, chimerism was demonstrated to be present in germ-line tissues, an event never before documented as naturally occurring in a primate. In fact, we found that chimeric marmosets often transmit sibling alleles acquired in utero to their own offspring. Thus, an individual that contributes gametes to an offspring is not necessarily the genetic parent of that offspring. The presence of somatic and germ-line chimerism may have influenced the evolution of the extensive paternal and alloparental care system of this taxon. Although the exact mechanisms of sociobiological change associated with chimerism have not been fully explored, we show here that chimerism alters relatedness between twins and may alter the perceived relatedness between family members, thus influencing the allocation of parental care. Consistent with this prediction, we found a significant correlation between paternal care effort and the presence of epithelial chimerism, with males carrying chimeric infants more often than nonchimeric infants. Therefore, we propose that the presence of placental chorionic fusion and the exchange of cell lines between embryos may represent a unique adaptation affecting the evolution of cooperative care in this group of primates.

  18. Thionin-D4E1 chimeric protein protects plants against bacterial infections

    DOEpatents

    Stover, Eddie W; Gupta, Goutam; Hao, Guixia

    2017-08-08

    The generation of a chimeric protein containing a first domain encoding either a pro-thionon or thionin, a second domain encoding D4E1 or pro-D4E1, and a third domain encoding a peptide linker located between the first domain and second domain is described. Either the first domain or the second domain is located at the amino terminal of the chimeric protein and the other domain (second domain or first domain, respectively) is located at the carboxyl terminal. The chimeric protein has antibacterial activity. Genetically altered plants and their progeny expressing a polynucleotide encoding the chimeric protein resist diseases caused by bacteria.

  19. Mediators of homologous DNA pairing.

    PubMed

    Zelensky, Alex; Kanaar, Roland; Wyman, Claire

    2014-10-09

    Homologous DNA pairing and strand exchange are at the core of homologous recombination. These reactions are promoted by a DNA-strand-exchange protein assembled into a nucleoprotein filament comprising the DNA-pairing protein, ATP, and single-stranded DNA. The catalytic activity of this molecular machine depends on control of its dynamic instability by accessory factors. Here we discuss proteins known as recombination mediators that facilitate formation and functional activation of the DNA-strand-exchange protein filament. Although the basics of homologous pairing and DNA-strand exchange are highly conserved in evolution, differences in mediator function are required to cope with differences in how single-stranded DNA is packaged by the single-stranded DNA-binding protein in different species, and the biochemical details of how the different DNA-strand-exchange proteins nucleate and extend into a nucleoprotein filament. The set of (potential) mediator proteins has apparently expanded greatly in evolution, raising interesting questions about the need for additional control and coordination of homologous recombination in more complex organisms. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  20. Mediators of Homologous DNA Pairing

    PubMed Central

    Zelensky, Alex; Kanaar, Roland; Wyman, Claire

    2014-01-01

    Homologous DNA pairing and strand exchange are at the core of homologous recombination. These reactions are promoted by a DNA-strand-exchange protein assembled into a nucleoprotein filament comprising the DNA-pairing protein, ATP, and single-stranded DNA. The catalytic activity of this molecular machine depends on control of its dynamic instability by accessory factors. Here we discuss proteins known as recombination mediators that facilitate formation and functional activation of the DNA-strand-exchange protein filament. Although the basics of homologous pairing and DNA-strand exchange are highly conserved in evolution, differences in mediator function are required to cope with differences in how single-stranded DNA is packaged by the single-stranded DNA-binding protein in different species, and the biochemical details of how the different DNA-strand-exchange proteins nucleate and extend into a nucleoprotein filament. The set of (potential) mediator proteins has apparently expanded greatly in evolution, raising interesting questions about the need for additional control and coordination of homologous recombination in more complex organisms. PMID:25301930

  1. Intertypic modular exchanges of genomic segments by homologous recombination at universally conserved segments in human adenovirus species D.

    PubMed

    Gonzalez, Gabriel; Koyanagi, Kanako O; Aoki, Koki; Kitaichi, Nobuyoshi; Ohno, Shigeaki; Kaneko, Hisatoshi; Ishida, Susumu; Watanabe, Hidemi

    2014-08-15

    Human adenovirus species D (HAdV-D), which is composed of clinically and epidemiologically important pathogens worldwide, contains more taxonomic "types" than any other species of the genus Mastadenovirus, although the mechanisms accounting for the high level of diversity remain to be disclosed. Recent studies of known and new types of HAdV-D have indicated that intertypic recombination between distant types contributes to the increasing diversity of the species. However, such findings raise the question as to how homologous recombination events occur between diversified types since homologous recombination is suppressed as nucleotide sequences diverge. In order to address this question, we investigated the distribution of the recombination boundaries in comparison with the landscape of intergenomic sequence conservation assessed according to the synonymous substitution rate (dS). The results revealed that specific genomic segments are conserved between even the most distantly related genomes; we call these segments "universally conserved segments" (UCSs). These findings suggest that UCSs facilitate homologous recombination, resulting in intergenomic segmental exchanges of UCS-flanking genomic regions as recombination modules. With the aid of such a mechanism, the haploid genomes of HAdV-Ds may have been reshuffled, resulting in chimeric genomes out of diversified repertoires in the HAdV-D population analogous to the MHC region reshuffled via crossing over in vertebrates. In addition, some HAdVs with chimeric genomes may have had the opportunity to avoid host immune responses thereby causing epidemics. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Novel nanocomposites from spider silk–silica fusion (chimeric) proteins

    PubMed Central

    Wong Po Foo, Cheryl; Patwardhan, Siddharth V.; Belton, David J.; Kitchel, Brandon; Anastasiades, Daphne; Huang, Jia; Naik, Rajesh R.; Perry, Carole C.; Kaplan, David L.

    2006-01-01

    Silica skeletal architectures in diatoms are characterized by remarkable morphological and nanostructural details. Silk proteins from spiders and silkworms form strong and intricate self-assembling fibrous biomaterials in nature. We combined the features of silk with biosilica through the design, synthesis, and characterization of a novel family of chimeric proteins for subsequent use in model materials forming reactions. The domains from the major ampullate spidroin 1 (MaSp1) protein of Nephila clavipes spider dragline silk provide control over structural and morphological details because it can be self-assembled through diverse processing methods including film casting and fiber electrospinning. Biosilica nanostructures in diatoms are formed in aqueous ambient conditions at neutral pH and low temperatures. The R5 peptide derived from the silaffin protein of Cylindrotheca fusiformis induces and regulates silica precipitation in the chimeric protein designs under similar ambient conditions. Whereas mineralization reactions performed in the presence of R5 peptide alone form silica particles with a size distribution of 0.5–10 μm in diameter, reactions performed in the presence of the new fusion proteins generate nanocomposite materials containing silica particles with a narrower size distribution of 0.5–2 μm in diameter. Furthermore, we demonstrate that composite morphology and structure could be regulated by controlling processing conditions to produce films and fibers. These results suggest that the chimeric protein provides new options for processing and control over silica particle sizes, important benefits for biomedical and specialty materials, particularly in light of the all aqueous processing and the nanocomposite features of these new materials. PMID:16769898

  3. Novel nanocomposites from spider silk-silica fusion (chimeric) proteins.

    PubMed

    Wong Po Foo, Cheryl; Patwardhan, Siddharth V; Belton, David J; Kitchel, Brandon; Anastasiades, Daphne; Huang, Jia; Naik, Rajesh R; Perry, Carole C; Kaplan, David L

    2006-06-20

    Silica skeletal architectures in diatoms are characterized by remarkable morphological and nanostructural details. Silk proteins from spiders and silkworms form strong and intricate self-assembling fibrous biomaterials in nature. We combined the features of silk with biosilica through the design, synthesis, and characterization of a novel family of chimeric proteins for subsequent use in model materials forming reactions. The domains from the major ampullate spidroin 1 (MaSp1) protein of Nephila clavipes spider dragline silk provide control over structural and morphological details because it can be self-assembled through diverse processing methods including film casting and fiber electrospinning. Biosilica nanostructures in diatoms are formed in aqueous ambient conditions at neutral pH and low temperatures. The R5 peptide derived from the silaffin protein of Cylindrotheca fusiformis induces and regulates silica precipitation in the chimeric protein designs under similar ambient conditions. Whereas mineralization reactions performed in the presence of R5 peptide alone form silica particles with a size distribution of 0.5-10 microm in diameter, reactions performed in the presence of the new fusion proteins generate nanocomposite materials containing silica particles with a narrower size distribution of 0.5-2 microm in diameter. Furthermore, we demonstrate that composite morphology and structure could be regulated by controlling processing conditions to produce films and fibers. These results suggest that the chimeric protein provides new options for processing and control over silica particle sizes, important benefits for biomedical and specialty materials, particularly in light of the all aqueous processing and the nanocomposite features of these new materials.

  4. Characterization of chimeric Bacillus thuringiensis Vip3 toxins.

    PubMed

    Fang, Jun; Xu, Xiaoli; Wang, Ping; Zhao, Jian-Zhou; Shelton, Anthony M; Cheng, Jiaan; Feng, Ming-Guang; Shen, Zhicheng

    2007-02-01

    Bacillus thuringiensis vegetative insecticidal proteins (Vip) are potential alternatives for B. thuringiensis endotoxins that are currently utilized in commercial transgenic insect-resistant crops. Screening a large number of B. thuringiensis isolates resulted in the cloning of vip3Ac1. Vip3Ac1 showed high insecticidal activity against the fall armyworm Spodoptera frugiperda and the cotton bollworm Helicoverpa zea but very low activity against the silkworm Bombyx mori. The host specificity of this Vip3 toxin was altered by sequence swapping with a previously identified toxin, Vip3Aa1. While both Vip3Aa1 and Vip3Ac1 showed no detectable toxicity against the European corn borer Ostrinia nubilalis, the chimeric protein Vip3AcAa, consisting of the N-terminal region of Vip3Ac1 and the C-terminal region of Vip3Aa1, became insecticidal to the European corn borer. In addition, the chimeric Vip3AcAa had increased toxicity to the fall armyworm. Furthermore, both Vip3Ac1 and Vip3AcAa are highly insecticidal to a strain of cabbage looper (Trichoplusia ni) that is highly resistant to the B. thuringiensis endotoxin Cry1Ac, thus experimentally showing for the first time the lack of cross-resistance between B. thuringiensis Cry1A proteins and Vip3A toxins. The results in this study demonstrated that vip3Ac1 and its chimeric vip3 genes can be excellent candidates for engineering a new generation of transgenic plants for insect pest control.

  5. Chimeric antibodies with extended half-life in ferrets.

    PubMed

    Nesspor, Thomas C; Scallon, Bernard

    2014-09-01

    Ferrets have long been used as a disease model for the study of influenza vaccines, but a more recent use has been for the study of human monoclonal antibodies directed against influenza viruses. Published data suggest that human antibodies are cleared unusually quickly from the ferret and that immune responses may be partially responsible. This immunogenicity increases variability within groups and may present an obstacle to long-term studies. Our aim was to identify an antibody design with reduced immunogenicity and longer circulating half-life in ferrets. The constant region coding sequences for ferret immunoglobulin G were cloned, and chimeric human/ferret antibodies were expressed and purified. Some of the chimeric antibodies included substitutions that have been shown to extend the half-life of human IgG antibodies. These chimeric antibodies were tested for binding to recombinant ferret FcRn receptor and then evaluated in pharmacokinetic studies in ferrets. A one-residue substitution in the ferret Fc domain, S252Y, was identified that increased binding affinity to the ferret neonatal receptor by 24-fold and extended half-life from 65 ± 27 to 206 ± 28 hours or ~9 days. Ferrets dosed twice with this surrogate antibody showed no indications of an immune response. Expressing the variable region of a candidate human therapeutic antibody with ferret constant regions containing the S252Y substitution can offer long half-life and limit immunogenicity. © 2014 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

  6. Chimeras taking shape: Potential functions of proteins encoded by chimeric RNA transcripts

    PubMed Central

    Frenkel-Morgenstern, Milana; Lacroix, Vincent; Ezkurdia, Iakes; Levin, Yishai; Gabashvili, Alexandra; Prilusky, Jaime; del Pozo, Angela; Tress, Michael; Johnson, Rory; Guigo, Roderic; Valencia, Alfonso

    2012-01-01

    Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans. PMID:22588898

  7. Bioengineered Chimeric Spider Silk-Uranium Binding Proteins

    PubMed Central

    Krishnaji, Sreevidhya Tarakkad; Kaplan, David L.

    2014-01-01

    Heavy metals constitute a source of environmental pollution. Here, novel functional hybrid biomaterials for specific interactions with heavy metals are designed by bioengineering consensus sequence repeats from spider silk of Nephila clavipes with repeats of a uranium peptide recognition motif from a mutated 33-residue of calmodulin protein from Paramecium tetraurelia. The self-assembly features of the silk to control nanoscale organic/inorganic material interfaces provides new biomaterials for uranium recovery. With subsequent enzymatic digestion of the silk to concentrate the sequestered metals, options can be envisaged to use these new chimeric protein systems in environmental engineering, including to remediate environments contaminated by uranium. PMID:23212989

  8. Chimeric transcripts resulting from complex duplications in chromosome Xq28.

    PubMed

    Zuccherato, Luciana W; Alleva, Benjamin; Whiters, Marjorie A; Carvalho, Claudia M B; Lupski, James R

    2016-02-01

    Gene fusions have been observed in somatic alterations in cancer and in schizophrenia. However, the underlying mechanism(s) for their formation are poorly understood. We experimentally demonstrated the expression of splicing variants of in silico predicted chimeric genes F8/CSAG1 and BCAP31/TEX28 in two individuals with de novo complex genomic rearrangements of Xq28; F8/CSAG1 includes exonization of an ERVL-MaLR intronic repetitive element. We provide evidence that replicative repair may contribute to exon shuffling processes and diversify the repertoire of expressed transcripts.

  9. The basic principles of chimeric antigen receptor (CAR) design

    PubMed Central

    Sadelain, Michel; Brentjens, Renier; Riviere, Isabelle

    2013-01-01

    CARs are recombinant receptors that provide both antigen-binding and T cell activating functions. A multitude of CARs has been reported over the past decade, targeting an array of cell surface tumor antigens. Their biological functions have dramatically changed following the introduction of tri-partite receptors comprising a costimulatory domain, termed second generation CARs. These have recently demonstrated clinical benefit in patients treated with CD19-targeted autologous T cells. CARs may be combined with costimulatory ligands, chimeric costimulatory receptors or cytokines to further enhance T cell potency, specificity and safety. CARs represent a new class of drugs with exciting potential for cancer immunotherapy. PMID:23550147

  10. Restriction-Stimulated Homologous Recombination of Plasmids by the Rece Pathway of Escherichia Coli

    PubMed Central

    Nussbaum, A.; Shalit, M.; Cohen, A.

    1992-01-01

    To test the double-strand break (DSB) repair model in recombination by the RecE pathway of Escherichia coli, we constructed chimeric phages that allow restriction-mediated release of linear plasmid substrates of the bioluminescence recombination assay in infected EcoRI(+) cells. Kinetics of DSB repair and expression of recombination products were followed by Southern hybridization and by the bioluminescence recombination assay, respectively. Plasmid recombinants were analyzed with restriction endonucleases. Our results indicate that a DSB can induce more than one type of RecE-mediated recombination. A DSB within the homology induced intermolecular recombination that followed the rules of the DSB repair model: (1) Recombination was enhanced by in vivo restriction. (2) Repair of the break depended on homologous sequences on the resident plasmid. (3) Break-repair was frequently associated with conversion of alleles that were cis to the break. (4) Conversion frequency decreased as the distance from the break increased. (5) Some clones contained a mixture of plasmid recombinants as expected by replication of a heteroduplex in the primary recombinant. The rules of the DSB repair model were not followed when recombination was induced by a DSB outside the homology. Both the cut and the uncut substrates were recipients in conversion events. Recombination events were associated with deletions that spanned the break site, but these deletions did not reach the homology. We propose that a break outside the homology may stimulate a RecE-mediated recombination pathway that does not involve direct participation of DNA ends in the homologous pairing reaction. PMID:1732167

  11. Detection of homologous blood transfusion.

    PubMed

    Voss, S C; Thevis, M; Schinkothe, T; Schänzer, W

    2007-08-01

    The aim of the present study was to improve and validate a flow cytometric method for the detection of homologous blood transfusion in doping control analysis. A panel of eight different primary antibodies and two different phycoerythrin-conjugated secondary antibodies was used for the detection of different blood populations. The flow cytometer used in this study was the BD FACSArray instrument. Mixed red blood cell populations were prepared from phenotype known donors. Linearity, specificity, recovery, precision, robustness and interday-precision were tested for every primary antibody used in the presented assay. The technique of signal amplification was utilized for an improved separation of antigens with weak or heterozygous expression to improve the interpretation of histograms. The resulting method allowed to clearly identify mixed red blood cell populations in homologous blood transfusion samples containing 0.3 - 2.0 % of donor blood.

  12. Chimeric honeybees (Apis mellifera) produced by transplantation of embryonic cells into pre-gastrula stage embryos and detection of chimerism by use of microsatellite markers.

    PubMed

    Bergem, M; Norberg, K; Roseth, A; Meuwissen, T; Lien, S; Aamodt, R H

    2006-04-01

    The production of chimeras, by use of cell transplantation, has proved to be highly valuable in studies of development by providing insights into cell fate, differentiation, and developmental potential. So far, chimeric honeybees have been created by nuclear transfer technologies. We have developed protocols to produce chimeric honeybees by use of cell transplantation. Embryonic cells were transplanted between pre-gastrula stage embryos (32-34 hr after oviposition) and hatched larvae were reared in vitro for 4 days. Chimeric individuals were detected by use of microsatellite analysis and a conservative estimation approach. 4.8% of embryos, posteriorly injected with embryonic cells, developed into chimeric honeybee larvae. By injection of cells pre-stained with fluorescent cell tracer dye, we studied the integration of transplanted cells in the developing embryos. Number of injected cells varied from 0 to 50 and cells remained and multiplied mainly in the area of injection.

  13. Homologies in Physics and Astrophysics

    NASA Astrophysics Data System (ADS)

    Bartlett, David F.; Cumalat, J. P.

    2012-01-01

    The genes of humans and chimpanzees are homologs. These genes are - in large measure - identical. From this detailed observation, we naturally suppose that both species evolved from a common ancestor. In particle physics the ordinary observed particles and their superymmetric partners are thought to be homologs, generated by a common "ancestor” , the Higgs particle. Experiments at CERN currently are testing this comfortable analogy of physics with biology. Neither the Higgs boson nor any supersymmetric particle has yet been found. We speculate that a variety of objects are homologs - evidence of an as yet undeveloped quantum theory of gravity to replace Dark Matter. A purely astronomical homology is the Vc - σ o relation which places nearly spherical elliptical galaxies just above well-formed spirals (SA & SB). Here the asymptotically- flat, circular velocity Vc is observed to be between 1 and 2 times the central bulge velocity dispersion σo over the range 60 km/s< σo <400 km/s (Ferrarese 2002, Fig 3). The Vc - σ o relation is difficult to explain with self-consistent equilibrium galaxy models (Courteau et al 2007). Here we give an explanation based on the Sinusoidal Potential, a non-Newtonian potential in which φ =-GM Cos[ko r]/r and ko=2 π /400 pc. We relate the lower limit of 60 km/s to the thermal velocity of protons at the” Broadhurst/Hirano & Hartnett” lookback redshift Z=105.6. This is the redshift where what was 400 pc then expands to 128 h-1 Mpc today. Further, at this Z the temperature of the universe was close to the Hartree Energy of 2 times 13.6 eV, an energy where protons have an rms speed of about 60 km/s.

  14. Chimeric Genes as a Source of Rapid Evolution in Drosophila melanogaster

    PubMed Central

    Rogers, Rebekah L.; Hartl, Daniel L.

    2012-01-01

    Chimeric genes form through the combination of portions of existing coding sequences to create a new open reading frame. These new genes can create novel protein structures that are likely to serve as a strong source of novelty upon which selection can act. We have identified 14 chimeric genes that formed through DNA-level mutations in Drosophila melanogaster, and we investigate expression profiles, domain structures, and population genetics for each of these genes to examine their potential to effect adaptive evolution. We find that chimeric gene formation commonly produces mid-domain breaks and unites portions of wholly unrelated peptides, creating novel protein structures that are entirely distinct from other constructs in the genome. These new genes are often involved in selective sweeps. We further find a disparity between chimeric genes that have recently formed and swept to fixation versus chimeric genes that have been preserved over long periods of time, suggesting that preservation and adaptation are distinct processes. Finally, we demonstrate that chimeric gene formation can produce qualitative expression changes that are difficult to mimic through duplicate gene formation, and that extremely young chimeric genes (dS < 0.03) are more likely to be associated with selective sweeps than duplicate genes of the same age. Hence, chimeric genes can serve as an exceptional source of genetic novelty that can have a profound influence on adaptive evolution in D. melanogaster. PMID:21771717

  15. Deep homology: a view from systematics.

    PubMed

    Scotland, Robert W

    2010-05-01

    Over the past decade, it has been discovered that disparate aspects of morphology - often of distantly related groups of organisms - are regulated by the same genetic regulatory mechanisms. Those discoveries provide a new perspective on morphological evolutionary change. A conceptual framework for exploring these research findings is termed 'deep homology'. A comparative framework for morphological relations of homology is provided that distinguishes analogy, homoplasy, plesiomorphy and synapomorphy. Four examples - three from plants and one from animals - demonstrate that homologous developmental mechanisms can regulate a range of morphological relations including analogy, homoplasy and examples of uncertain homology. Deep homology is part of a much wider range of phenomena in which biological (genes, regulatory mechanisms, morphological traits) and phylogenetic levels of homology can both be disassociated. Therefore, to understand homology, precise, comparative, independent statements of both biological and phylogenetic levels of homology are necessary.

  16. Chimeric antigen receptor engineered stem cells: a novel HIV therapy.

    PubMed

    Zhen, Anjie; Carrillo, Mayra A; Kitchen, Scott G

    2017-03-01

    Despite the success of combination antiretroviral therapy (cART) for suppressing HIV and improving patients' quality of life, HIV persists in cART-treated patients and remains an incurable disease. Financial burdens and health consequences of lifelong cART treatment call for novel HIV therapies that result in a permanent cure. Cellular immunity is central in controlling HIV replication. However, HIV adopts numerous strategies to evade immune surveillance. Engineered immunity via genetic manipulation could offer a functional cure by generating cells that have enhanced antiviral activity and are resistant to HIV infection. Recently, encouraging reports from several human clinical trials using an anti-CD19 chimeric antigen receptor (CAR) modified T-cell therapy for treating B-cell malignancies have provided valuable insights and generated remarkable enthusiasm in engineered T-cell therapy. In this review, we discuss the development of HIV-specific chimeric antigen receptors and the use of stem cell based therapies to generate lifelong anti-HIV immunity.

  17. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ].

    PubMed

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P

    2015-01-01

    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  18. Chimeric behavior of excited thioxanthone in protic solvents: II. Theory.

    PubMed

    Rai-Constapel, Vidisha; Villnow, Torben; Ryseck, Gerald; Gilch, Peter; Marian, Christel M

    2014-12-18

    The chimeric behavior of thioxanthone in protic solvents has been investigated employing computational chemistry methods. In particular, methanol and 2,2,2-trifluoroethanol have been chosen in this study. The solvent environment has been modeled using microsolvation in combination with a conductor-like screening model. The vertical excitation spectrum within the same solvent is seen to depend on the number of specific bonds formed between the chromophore and the solvent molecules. Two different models have been discussed in this work, namely, one and two H-bond models. In particular, the formation of the second H-bond causes the energy gap between the πHπL* and nOπL* states to increase further. Excited-state absorption spectra for the photophysically relevant electronic states have been theoretically determined for comparison with the time-resolved spectra recorded experimentally [Villnow, T.; Ryseck, G.; Rai-Constapel, V.; Marian, C. M.; Gilch, P. J. Phys. Chem. A 2014]. The equilibration of the 1(πHπL*) and 3(nOπL*) states holds responsible for the chimeric behavior. This equilibrium sets in with a calculated time constant of 23 ps in methanol and 14 ps in TFE (5 and 10 ps in experiment, respectively). The radiative decay from the optically bright 1(πHπL*) state is computed to occur with a time constant of 25 ns in both solvents (14–25 ns in experiment).

  19. Preliminary analgesic properties of deltorphin-5-methoxytryptamine chimeric opioid peptides.

    PubMed

    Wang, Jing; Wang, Li; Li, Meixing; Jin, Qiaoying; Dong, Shouliang

    2011-05-01

    To further understand the relationship between melatonin (MT) and deltorphins (Dels) in pain modulation, two chimeric peptides (Del I-5-methoxytryptamine and Del II-5-methoxytryptamine) both containing 5-methoxytryptamine at the carboxyl-terminal of Dels mimicking MT were designed, synthesized and characterized by tail-flick assay in mice. Results showed that intracerebroventricular (i.c.v.) administration of Del I-5-methoxytryptamine (YaFDVVG-X, X is 5-methoxytryptamine, 5, 50 nmol/kg) or Del II-5-methoxytryptamine (YaFEVVG-X, X is 5-methoxytryptamine, 5, 50 nmol/kg) produced stronger analgesia than deltorphins (Del I or Del II alone), and acting even longer and stronger than cocktails containing Del I or Del II (50 nmol/kg) and MT (50 nmol/kg). Naloxone (i.p., 100 nmol/kg) could totally block the analgesic effects induced by the chimeric peptides, while luzindole (specific antagonist of melatonin receptor, i.p., 250 nmol/kg) could only partially inhibit the effects down to that induced by Dels alone. Interestingly, Del I-5-methoxytryptamine and Del II-5-methoxytryptamine act weaker with δ receptor than Dels in vitro but could induce much longer analgesia through co-activating δ opioid receptor and melatonin receptor.

  20. Mixed chimerism to induce tolerance for solid organ transplantation

    SciTech Connect

    Wren, S.M.; Nalesnik, M.; Hronakes, M.L.; Oh, E.; Ildstad, S.T. )

    1991-04-01

    Chimerism, or the coexistence of tissue elements from more than one genetically different strain or species in an organism, is the only experimental state that results in the induction of donor-specific transplantation tolerance. Transplantation of a mixture of T-cell-depleted syngeneic (host-type) plus T-cell-depleted allogeneic (donor) bone marrow into a normal adult recipient mouse (A + B----A) results in mixed allogeneic chimerism. Recipient mice exhibit donor-specific transplantation tolerance, yet have full immunocompetence to recognize and respond to third-party transplantation antigens. After complete hematolymphopoietic repopulation at 28 days, animals accept a donor-specific skin graft but reject major histocompatibility complex (MHC) locus-disparate third-party grafts. We now report that permanent graft acceptance can also be achieved when the graft is placed at the time of bone marrow transplantation. Histologically, grafts were viable and had only minimal inflammatory changes. This model may have potential future clinical application for the induction of donor-specific transplantation tolerance.

  1. Chimeric Antisense Oligonucleotide Conjugated to α-Tocopherol

    PubMed Central

    Nishina, Tomoko; Numata, Junna; Nishina, Kazutaka; Yoshida-Tanaka, Kie; Nitta, Keiko; Piao, Wenying; Iwata, Rintaro; Ito, Shingo; Kuwahara, Hiroya; Wada, Takeshi; Mizusawa, Hidehiro; Yokota, Takanori

    2015-01-01

    We developed an efficient system for delivering short interfering RNA (siRNA) to the liver by using α-tocopherol conjugation. The α-tocopherol–conjugated siRNA was effective and safe for RNA interference–mediated gene silencing in vivo. In contrast, when the 13-mer LNA (locked nucleic acid)-DNA gapmer antisense oligonucleotide (ASO) was directly conjugated with α-tocopherol it showed markedly reduced silencing activity in mouse liver. Here, therefore, we tried to extend the 5′-end of the ASO sequence by using 5′-α-tocopherol–conjugated 4- to 7-mers of unlocked nucleic acid (UNA) as a “second wing.” Intravenous injection of mice with this α-tocopherol–conjugated chimeric ASO achieved more potent silencing than ASO alone in the liver, suggesting increased delivery of the ASO to the liver. Within the cells, the UNA wing was cleaved or degraded and α-tocopherol was released from the 13-mer gapmer ASO, resulting in activation of the gapmer. The α-tocopherol–conjugated chimeric ASO showed high efficacy, with hepatic tropism, and was effective and safe for gene silencing in vivo. We have thus identified a new, effective LNA-DNA gapmer structure in which drug delivery system (DDS) molecules are bound to ASO with UNA sequences. PMID:25584900

  2. CHIMERE-2017: from urban to hemispheric chemistry-transport modeling

    NASA Astrophysics Data System (ADS)

    Mailler, Sylvain; Menut, Laurent; Khvorostyanov, Dmitry; Valari, Myrto; Couvidat, Florian; Siour, Guillaume; Turquety, Solène; Briant, Régis; Tuccella, Paolo; Bessagnet, Bertrand; Colette, Augustin; Létinois, Laurent; Markakis, Kostantinos; Meleux, Frédérik

    2017-06-01

    CHIMERE is a chemistry-transport model designed for regional atmospheric composition. It can be used at a variety of scales from local to continental domains. However, due to the model design and its historical use as a regional model, major limitations had remained, hampering its use at hemispheric scale, due to the coordinate system used for transport as well as to missing processes that are important in regions outside Europe. Most of these limitations have been removed in the CHIMERE-2017 version, allowing its use in any region of the world and at any scale, from the scale of a single urban area up to hemispheric scale, with or without polar regions included. Other important improvements have been made in the treatment of the physical processes affecting aerosols and the emissions of mineral dust. From a computational point of view, the parallelization strategy of the model has also been updated in order to improve model numerical performance and reduce the code complexity. The present article describes all these changes. Statistical scores for a model simulation over continental Europe are presented, and a simulation of the circumpolar transport of volcanic ash plume from the Puyehue volcanic eruption in June 2011 in Chile provides a test case for the new model version at hemispheric scale.

  3. Gene targeting in the red alga Cyanidioschyzon merolae: single- and multi-copy insertion using authentic and chimeric selection markers.

    PubMed

    Fujiwara, Takayuki; Ohnuma, Mio; Yoshida, Masaki; Kuroiwa, Tsuneyoshi; Hirano, Tatsuya

    2013-01-01

    The unicellular red alga Cyanidioschyzon merolae is an emerging model organism for studying organelle division and inheritance: the cell is composed of an extremely simple set of organelles (one nucleus, one mitochondrion and one chloroplast), and their genomes are completely sequenced. Although a fruitful set of cytological and biochemical methods have now been developed, gene targeting techniques remain to be fully established in this organism. Thus far, only a single selection marker, URA Cm-Gs , has been available that complements the uracil-auxotrophic mutant M4. URA Cm-Gs , a chimeric URA5.3 gene of C. merolae and the related alga Galdieria sulphuraria, was originally designed to avoid gene conversion of the mutated URA5.3 allele in the parental strain M4. Although an early example of targeted gene disruption by homologous recombination was reported using this marker, the genome structure of the resultant transformants had never been fully characterized. In the current study, we showed that the use of the chimeric URA Cm-Gs selection marker caused multicopy insertion at high frequencies, accompanied by undesired recombination events at the targeted loci. The copy number of the inserted fragments was variable among the transformants, resulting in high yet uneven levels of transgene expression. In striking contrast, when the authentic URA5.3 gene (URA Cm-Cm ) was used as a selection marker, efficient single-copy insertion was observed at the targeted locus. Thus, we have successfully established a highly reliable and reproducible method for gene targeting in C. merolae. Our method will be applicable to a number of genetic manipulations in this organism, including targeted gene disruption, replacement and tagging.

  4. Structural basis for drug and substrate specificity exhibited by FIV encoding a chimeric FIV/HIV protease

    SciTech Connect

    Lin, Ying-Chuan; Perryman, Alexander L.; Olson, Arthur J.; Torbett, Bruce E.; Elder, John H.; Stout, C. David

    2011-06-01

    Crystal structures of the 6s-98S FIV protease chimera with darunavir and lopinavir bound have been determined at 1.7 and 1.8 Å resolution, respectively. A chimeric feline immunodeficiency virus (FIV) protease (PR) has been engineered that supports infectivity but confers sensitivity to the human immunodeficiency virus (HIV) PR inhibitors darunavir (DRV) and lopinavir (LPV). The 6s-98S PR has five replacements mimicking homologous residues in HIV PR and a sixth which mutated from Pro to Ser during selection. Crystal structures of the 6s-98S FIV PR chimera with DRV and LPV bound have been determined at 1.7 and 1.8 Å resolution, respectively. The structures reveal the role of a flexible 90s loop and residue 98 in supporting Gag processing and infectivity and the roles of residue 37 in the active site and residues 55, 57 and 59 in the flap in conferring the ability to specifically recognize HIV PR drugs. Specifically, Ile37Val preserves tertiary structure but prevents steric clashes with DRV and LPV. Asn55Met and Val59Ile induce a distinct kink in the flap and a new hydrogen bond to DRV. Ile98Pro→Ser and Pro100Asn increase 90s loop flexibility, Gln99Val contributes hydrophobic contacts to DRV and LPV, and Pro100Asn forms compensatory hydrogen bonds. The chimeric PR exhibits a comparable number of hydrogen bonds, electrostatic interactions and hydrophobic contacts with DRV and LPV as in the corresponding HIV PR complexes, consistent with IC{sub 50} values in the nanomolar range.

  5. Gene Targeting in the Red Alga Cyanidioschyzon merolae: Single- and Multi-Copy Insertion Using Authentic and Chimeric Selection Markers

    PubMed Central

    Fujiwara, Takayuki; Ohnuma, Mio; Yoshida, Masaki; Kuroiwa, Tsuneyoshi; Hirano, Tatsuya

    2013-01-01

    The unicellular red alga Cyanidioschyzon merolae is an emerging model organism for studying organelle division and inheritance: the cell is composed of an extremely simple set of organelles (one nucleus, one mitochondrion and one chloroplast), and their genomes are completely sequenced. Although a fruitful set of cytological and biochemical methods have now been developed, gene targeting techniques remain to be fully established in this organism. Thus far, only a single selection marker, URACm-Gs, has been available that complements the uracil-auxotrophic mutant M4. URACm-Gs, a chimeric URA5.3 gene of C. merolae and the related alga Galdieria sulphuraria, was originally designed to avoid gene conversion of the mutated URA5.3 allele in the parental strain M4. Although an early example of targeted gene disruption by homologous recombination was reported using this marker, the genome structure of the resultant transformants had never been fully characterized. In the current study, we showed that the use of the chimeric URACm-Gs selection marker caused multicopy insertion at high frequencies, accompanied by undesired recombination events at the targeted loci. The copy number of the inserted fragments was variable among the transformants, resulting in high yet uneven levels of transgene expression. In striking contrast, when the authentic URA5.3 gene (URACm-Cm) was used as a selection marker, efficient single-copy insertion was observed at the targeted locus. Thus, we have successfully established a highly reliable and reproducible method for gene targeting in C. merolae. Our method will be applicable to a number of genetic manipulations in this organism, including targeted gene disruption, replacement and tagging. PMID:24039997

  6. Vaccination with Vesicular Stomatitis Virus-Vectored Chimeric Hemagglutinins Protects Mice against Divergent Influenza Virus Challenge Strains.

    PubMed

    Ryder, Alex B; Nachbagauer, Raffael; Buonocore, Linda; Palese, Peter; Krammer, Florian; Rose, John K

    2015-12-16

    Seasonal influenza virus infections continue to cause significant disease each year, and there is a constant threat of the emergence of reassortant influenza strains causing a new pandemic. Available influenza vaccines are variably effective each season, are of limited scope at protecting against viruses that have undergone significant antigenic drift, and offer low protection against newly emergent pandemic strains. "Universal" influenza vaccine strategies that focus on the development of humoral immunity directed against the stalk domains of the viral hemagglutinin (HA) show promise for protecting against diverse influenza viruses. Here, we describe such a strategy that utilizes vesicular stomatitis virus (VSV) as a vector for chimeric hemagglutinin (cHA) antigens. This vaccination strategy is effective at generating HA stalk-specific, broadly cross-reactive serum antibodies by both intramuscular and intranasal routes of vaccination. We show that prime-boost vaccination strategies provide protection against both lethal homologous and heterosubtypic influenza challenge and that protection is significantly improved with intranasal vaccine administration. Additionally, we show that vaccination with VSV-cHAs generates greater stalk-specific and cross-reactive serum antibodies than does vaccination with VSV-vectored full-length HAs, confirming that cHA-based vaccination strategies are superior at generating stalk-specific humoral immunity. VSV-vectored influenza vaccines that express chimeric hemagglutinin antigens offer a novel means for protecting against widely diverging influenza viruses. Universal influenza vaccination strategies should be capable of protecting against a wide array of influenza viruses, and we have developed such an approach utilizing a single viral vector system. The potent antibody responses that these vaccines generate are shown to protect mice against lethal influenza challenges with highly divergent viruses. Notably, intranasal vaccination

  7. Donor Chimerism Early after Reduced-intensity Conditioning Hematopoietic Stem Cell Transplantation Predicts Relapse and Survival

    PubMed Central

    Koreth, John; Kim, Haesook T.; Nikiforow, Sarah; Milford, Edgar L.; Armand, Philippe; Cutler, Corey; Glotzbecker, Brett; Ho, Vincent T.; Antin, Joseph H.; Soiffer, Robert J.; Ritz, Jerome; Alyea, Edwin P.

    2015-01-01

    The impact of early donor cell chimerism on outcomes of T-replete reduced-intensity conditioning (RIC) hematopoietic stem cell transplantation (HSCT) is ill-defined. We evaluated day 30 (D30) and 100 (D100) total donor cell chimerism after RIC HSCT undertaken between 2002 and 2010 at our institution, excluding patients who died or relapsed before D30. When available, donor T-cell chimerism was also assessed. The primary outcome was overall survival (OS). Secondary outcomes included progression-free survival (PFS), relapse and non-relapse mortality (NRM). 688 patients with hematologic malignancies (48% myeloid; 52% lymphoid) and a median age of 57 years (range, 18-74) undergoing RIC HSCT with T-replete donor grafts (97% peripheral blood; 92% HLA-matched) and median follow-up of 58.2 months (range, 12.6-120.7) were evaluated. In multivariable analysis total donor cell and T-cell chimerism at D30 and D100 each predicted RIC HSCT outcomes, with D100 total donor cell chimerism most predictive. D100 total donor cell chimerism <90% was associated with increased relapse (HR 2.54, 95% CI 1.83-3.51, p<0.0001), impaired PFS (HR 2.01, 95% CI 1.53-2.65, p<0.0001) and worse OS (1.50, 95% CI 1.11-2.04, p=0.009), but not NRM (HR 0.76; 95% CI 0.44-2.27, p=0.33). There was no additional utility of incorporating sustained D30-D100 total donor cell chimerism, or T-cell chimerism. Low donor chimerism early after RIC HSCT is an independent risk factor for relapse and impaired survival. Donor chimerism assessment early after RIC HSCT can prognosticate for long-term outcomes and help identify high-risk patient cohorts that may benefit from additional therapeutic interventions. PMID:24907627

  8. Safety of targeting ROR1 in primates with chimeric antigen receptor-modified T cells

    PubMed Central

    Berger, Carolina; Sommermeyer, Daniel; Hudecek, Michael; Berger, Michael; Balakrishnan, Ashwini; Paszkiewicz, Paulina J.; Kosasih, Paula L.; Rader, Christoph; Riddell, Stanley R.

    2014-01-01

    Genetic engineering of T cells for adoptive transfer by introducing a tumor-targeting chimeric antigen receptor (CAR) is a new approach to cancer immunotherapy. A challenge for the field is to define cell surface molecules that are both preferentially expressed on tumor cells and can be safely targeted with T cells. The orphan tyrosine kinase receptor ROR1 is a candidate target for T-cell therapy with CAR-modified T cells (CAR-T cells) since it is expressed on the surface of many lymphatic and epithelial malignancies and has a putative role in tumor cell survival. The cell surface isoform of ROR1 is expressed in embryogenesis but absent in adult tissues except for B-cell precursors, and low levels of transcripts in adipocytes, pancreas, and lung. ROR1 is highly conserved between humans and macaques and has a similar pattern of tissue expression. To determine if low-level ROR1-expression on normal cells would result in toxicity or adversely affect CAR-T cell survival and/or function, we adoptively transferred autologous ROR1 CAR-T cells into nonhuman primates. ROR1 CAR-T cells did not cause overt toxicity to normal organs and accumulated in bone marrow and lymph node sites where ROR1-positive B cells were present. The findings support the clinical evaluation of ROR1 CAR-T cells for ROR1+ malignancies and demonstrate the utility of nonhuman primates for evaluating the safety of immunotherapy with engineered T cells specific for tumor-associated molecules that are homologous between humans and nonhuman primates. PMID:25355068

  9. Calcium-stimulated autophosphorylation site of plant chimeric calcium/calmodulin-dependent protein kinase

    NASA Technical Reports Server (NTRS)

    Sathyanarayanan, P. V.; Siems, W. F.; Jones, J. P.; Poovaiah, B. W.

    2001-01-01

    The existence of two molecular switches regulating plant chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK), namely the C-terminal visinin-like domain acting as Ca(2+)-sensitive molecular switch and calmodulin binding domain acting as Ca(2+)-stimulated autophosphorylation-sensitive molecular switch, has been described (Sathyanarayanan, P. V., Cremo, C. R., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 30417-30422). Here we report the identification of Ca(2+)-stimulated autophosphorylation site of CCaMK by matrix-assisted laser desorption ionization time of flight-mass spectrometry. Thr(267) was confirmed as the Ca(2+)-stimulated autophosphorylation site by post-source decay experiments and by site-directed mutagenesis. The purified T267A mutant form of CCaMK did not show Ca(2+)-stimulated autophosphorylation, autophosphorylation-dependent variable calmodulin affinity, or Ca(2+)/calmodulin stimulation of kinase activity. Sequence comparison of CCaMK from monocotyledonous plant (lily) and dicotyledonous plant (tobacco) suggests that the autophosphorylation site is conserved. This is the first identification of a phosphorylation site specifically responding to activation by second messenger system (Ca(2+) messenger system) in plants. Homology modeling of the kinase and calmodulin binding domain of CCaMK with the crystal structure of calcium/calmodulin-dependent protein kinase 1 suggests that the Ca(2+)-stimulated autophosphorylation site is located on the surface of the kinase and far from the catalytic site. Analysis of Ca(2+)-stimulated autophosphorylation with increasing concentration of CCaMK indicates the possibility that the Ca(2+)-stimulated phosphorylation occurs by an intermolecular mechanism.

  10. Calcium-stimulated autophosphorylation site of plant chimeric calcium/calmodulin-dependent protein kinase

    NASA Technical Reports Server (NTRS)

    Sathyanarayanan, P. V.; Siems, W. F.; Jones, J. P.; Poovaiah, B. W.

    2001-01-01

    The existence of two molecular switches regulating plant chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK), namely the C-terminal visinin-like domain acting as Ca(2+)-sensitive molecular switch and calmodulin binding domain acting as Ca(2+)-stimulated autophosphorylation-sensitive molecular switch, has been described (Sathyanarayanan, P. V., Cremo, C. R., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 30417-30422). Here we report the identification of Ca(2+)-stimulated autophosphorylation site of CCaMK by matrix-assisted laser desorption ionization time of flight-mass spectrometry. Thr(267) was confirmed as the Ca(2+)-stimulated autophosphorylation site by post-source decay experiments and by site-directed mutagenesis. The purified T267A mutant form of CCaMK did not show Ca(2+)-stimulated autophosphorylation, autophosphorylation-dependent variable calmodulin affinity, or Ca(2+)/calmodulin stimulation of kinase activity. Sequence comparison of CCaMK from monocotyledonous plant (lily) and dicotyledonous plant (tobacco) suggests that the autophosphorylation site is conserved. This is the first identification of a phosphorylation site specifically responding to activation by second messenger system (Ca(2+) messenger system) in plants. Homology modeling of the kinase and calmodulin binding domain of CCaMK with the crystal structure of calcium/calmodulin-dependent protein kinase 1 suggests that the Ca(2+)-stimulated autophosphorylation site is located on the surface of the kinase and far from the catalytic site. Analysis of Ca(2+)-stimulated autophosphorylation with increasing concentration of CCaMK indicates the possibility that the Ca(2+)-stimulated phosphorylation occurs by an intermolecular mechanism.

  11. Structure-based affinity maturation of a chimeric anti-ricin antibody C4C13.

    PubMed

    Luo, Longlong; Luo, Qun; Guo, Leiming; Lv, Ming; Lin, Zhou; Geng, Jing; Li, Xinying; Li, Yan; Shen, Beifen; Qiao, Chunxia; Feng, Jiannan

    2014-01-01

    Ricin is a highly lethal toxin. Anti-ricin chimeric monoclonal antibody (mAb) C4C13 was prepared in our lab; however, its binding affinity was much weaker than that of the parent antibody 4C13. In this study, based on the computer-guided homology modeling and conformational optimization methods, the 3-D structure of C4C13 variable regions Fv was constructed and optimized. Using molecular docking and dynamics simulation methods, the 3-D complex structure of ricin and C4C13 Fv was obtained. Considering the orientation property, surface electrostatic distribution, residues chemical and physical character and intermolecular hydrogen bond, the binding mode and key residues were predicted. According to C4C13 Fv fragment and ricin complementary binding surface, electrostatic attraction periphery and van der Waals interaction interface, three mutants (i.e., M1 (N(H102)F, W(H103)Y); M2 (W(H103)Y) and M3 (R(L90)G)) were designed, in which M1 and M2 were predicted to possess higher antigen-binding activity than C4C13, while M3 was weaker. The relative affinity assays by ELISA showed that M1 and M2 mutations had higher affinity (9.6 and 18.3 nmol/L) than C4C13 (130 nmol/L) and M3 had weaker affinity (234.5 nmol/L) than C4C13. The results showed that the modeling complex structure of the antigen (ricin) and antibody (C4C13) is reasonable. Our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-ricin antibody design and preparation in future.

  12. Definition and identification of homology domains.

    PubMed

    Lawrence, C B; Goldman, D A

    1988-03-01

    A method is described for identifying and evaluating regions of significant similarity between two sequences. The notion of a 'homology domain' is employed which defines the boundaries of a region of sequence homology containing no insertions or deletions. The relative significance of different potential homology domains is evaluated using a non-linear similarity score related to the probability of finding the observed level of similarity in the region by chance. The sensitivity of the method is demonstrated by simulating the evolution of homology domains and applying the method to their detection. Several examples of the use of homology domain identification are given.

  13. Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

    PubMed Central

    Whitaker, Neal; Berry, Trista M.; Rosenthal, Nathan; Gordon, Jay E.; Gonzalez-Rivera, Christian; Sheehan, Kathy B.; Truchan, Hilary K.; VieBrock, Lauren; Newton, Irene L. G.; Carlyon, Jason A.

    2016-01-01

    ABSTRACT Bacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis. A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4At) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4At and the TraJ/VirD4At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ΔCTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4At's CTD to the VirE2 effector, although other VirD4At domains bound this substrate in vitro. We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains. IMPORTANCE Bacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to

  14. Establishing homologies in protein sequences

    NASA Technical Reports Server (NTRS)

    Dayhoff, M. O.; Barker, W. C.; Hunt, L. T.

    1983-01-01

    Computer-based statistical techniques used to determine homologies between proteins occurring in different species are reviewed. The technique is based on comparison of two protein sequences, either by relating all segments of a given length in one sequence to all segments of the second or by finding the best alignment of the two sequences. Approaches discussed include selection using printed tabulations, identification of very similar sequences, and computer searches of a database. The use of the SEARCH, RELATE, and ALIGN programs (Dayhoff, 1979) is explained; sample data are presented in graphs, diagrams, and tables and the construction of scoring matrices is considered.

  15. Establishing homologies in protein sequences

    NASA Technical Reports Server (NTRS)

    Dayhoff, M. O.; Barker, W. C.; Hunt, L. T.

    1983-01-01

    Computer-based statistical techniques used to determine homologies between proteins occurring in different species are reviewed. The technique is based on comparison of two protein sequences, either by relating all segments of a given length in one sequence to all segments of the second or by finding the best alignment of the two sequences. Approaches discussed include selection using printed tabulations, identification of very similar sequences, and computer searches of a database. The use of the SEARCH, RELATE, and ALIGN programs (Dayhoff, 1979) is explained; sample data are presented in graphs, diagrams, and tables and the construction of scoring matrices is considered.

  16. Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia

    PubMed Central

    Maude, Shannon L.; Frey, Noelle; Shaw, Pamela A.; Aplenc, Richard; Barrett, David M.; Bunin, Nancy J.; Chew, Anne; Gonzalez, Vanessa E.; Zheng, Zhaohui; Lacey, Simon F.; Mahnke, Yolanda D.; Melenhorst, Jan J.; Rheingold, Susan R.; Shen, Angela; Teachey, David T.; Levine, Bruce L.; June, Carl H.; Porter, David L.; Grupp, Stephan A.

    2014-01-01

    BACKGROUND Relapsed acute lymphoblastic leukemia (ALL) is difficult to treat despite the availability of aggressive therapies. Chimeric antigen receptor–modified T cells targeting CD19 may overcome many limitations of conventional therapies and induce remission in patients with refractory disease. METHODS We infused autologous T cells transduced with a CD19-directed chimeric antigen receptor (CTL019) lentiviral vector in patients with relapsed or refractory ALL at doses of 0.76×106 to 20.6×106 CTL019 cells per kilogram of body weight. Patients were monitored for a response, toxic effects, and the expansion and persistence of circulating CTL019 T cells. RESULTS A total of 30 children and adults received CTL019. Complete remission was achieved in 27 patients (90%), including 2 patients with blinatumomab-refractory disease and 15 who had undergone stem-cell transplantation. CTL019 cells proliferated in vivo and were detectable in the blood, bone marrow, and cerebrospinal fluid of patients who had a response. Sustained remission was achieved with a 6-month event-free survival rate of 67% (95% confidence interval [CI], 51 to 88) and an overall survival rate of 78% (95% CI, 65 to 95). At 6 months, the probability that a patient would have persistence of CTL019 was 68% (95% CI, 50 to 92) and the probability that a patient would have relapse-free B-cell aplasia was 73% (95% CI, 57 to 94). All the patients had the cytokine-release syndrome. Severe cytokine-release syndrome, which developed in 27% of the patients, was associated with a higher disease burden before infusion and was effectively treated with the anti–interleukin-6 receptor antibody tocilizumab. CONCLUSIONS Chimeric antigen receptor–modified T-cell therapy against CD19 was effective in treating relapsed and refractory ALL. CTL019 was associated with a high remission rate, even among patients for whom stem-cell transplantation had failed, and durable remissions up to 24 months were observed. (Funded by

  17. Chimeric antigen receptor T cells for sustained remissions in leukemia.

    PubMed

    Maude, Shannon L; Frey, Noelle; Shaw, Pamela A; Aplenc, Richard; Barrett, David M; Bunin, Nancy J; Chew, Anne; Gonzalez, Vanessa E; Zheng, Zhaohui; Lacey, Simon F; Mahnke, Yolanda D; Melenhorst, Jan J; Rheingold, Susan R; Shen, Angela; Teachey, David T; Levine, Bruce L; June, Carl H; Porter, David L; Grupp, Stephan A

    2014-10-16

    Relapsed acute lymphoblastic leukemia (ALL) is difficult to treat despite the availability of aggressive therapies. Chimeric antigen receptor-modified T cells targeting CD19 may overcome many limitations of conventional therapies and induce remission in patients with refractory disease. We infused autologous T cells transduced with a CD19-directed chimeric antigen receptor (CTL019) lentiviral vector in patients with relapsed or refractory ALL at doses of 0.76×10(6) to 20.6×10(6) CTL019 cells per kilogram of body weight. Patients were monitored for a response, toxic effects, and the expansion and persistence of circulating CTL019 T cells. A total of 30 children and adults received CTL019. Complete remission was achieved in 27 patients (90%), including 2 patients with blinatumomab-refractory disease and 15 who had undergone stem-cell transplantation. CTL019 cells proliferated in vivo and were detectable in the blood, bone marrow, and cerebrospinal fluid of patients who had a response. Sustained remission was achieved with a 6-month event-free survival rate of 67% (95% confidence interval [CI], 51 to 88) and an overall survival rate of 78% (95% CI, 65 to 95). At 6 months, the probability that a patient would have persistence of CTL019 was 68% (95% CI, 50 to 92) and the probability that a patient would have relapse-free B-cell aplasia was 73% (95% CI, 57 to 94). All the patients had the cytokine-release syndrome. Severe cytokine-release syndrome, which developed in 27% of the patients, was associated with a higher disease burden before infusion and was effectively treated with the anti-interleukin-6 receptor antibody tocilizumab. Chimeric antigen receptor-modified T-cell therapy against CD19 was effective in treating relapsed and refractory ALL. CTL019 was associated with a high remission rate, even among patients for whom stem-cell transplantation had failed, and durable remissions up to 24 months were observed. (Funded by Novartis and others; CART19 Clinical

  18. A technical application of quantitative next generation sequencing for chimerism evaluation

    PubMed Central

    Aloisio, Michelangelo; Licastro, Danilo; Caenazzo, Luciana; Torboli, Valentina; D'eustacchio, Angela; Severini, Giovanni Maria; Athanasakis, Emmanouil

    2016-01-01

    At present, the most common genetic diagnostic method for chimerism evaluation following hematopoietic stem cell transplantation is microsatellite analysis by capillary electrophoresis. The main objective was to establish, through repeated analysis over time, if a complete chimerism was present, or if the mixed chimerism was stable, increasing or decreasing over time. Considering the recent introduction of next generation sequencing (NGS) in clinical diagnostics, a detailed study evaluating an NGS protocol was conducted, coupled with a custom bioinformatics pipeline, for chimerism quantification. Based on the technology of Ion AmpliSeq, a 44-amplicon custom chimerism panel was designed, and a custom bioinformatics pipeline dedicated to the genotyping and quantification of NGS data was coded. The custom chimerism panel allowed identification of an average of 16 informative recipient alleles. The limit of detection of the protocol was fixed at 1% due to the NGS background (<1%). The protocol followed the standard Ion AmpliSeq library preparation and Ion Torrent Personal Genome Machine guidelines. Overall, the present study added to the scientific literature, identifying novel technical details for a possible future application of NGS for chimerism quantification. PMID:27499173

  19. Structural homologies among the hemopoietins.

    PubMed Central

    Schrader, J W; Ziltener, H J; Leslie, K B

    1986-01-01

    A group of cytokines characterized by a common set of target cells--namely, the pluripotential hemopoietic stem cells or their cellular derivatives--share similarities in the amino acid sequence at their N terminus or in the putative signal peptide immediately prior to the published N terminus. Murine P-cell-stimulating factor (PSF), murine and human interleukin 2 (IL-2), murine and human granulocyte-macrophage colony-stimulating factor (GM-CSF), human erythropoietin, and human interleukin 1 beta all share alanine as the N-terminal amino acid and have some similarities in the succeeding three or four amino acids. In the case of murine PSF and GM-CSF, the six N-terminal amino acids are readily cleaved from mature molecules and are lacking from the N-terminal amino acid sequences reported initially. A sixth cytokine, colony-stimulating factor 1, has an alanine followed by a similar pattern of five amino acids at the end of the putative signal peptide. GM-CSF and IL-2 have more extensive homology, about 25% of residues being identical in three regions that comprise about 70% of the molecules. Only minor similarities of uncertain significance were found among the complete amino acid sequences of the other cytokines. Although its evolutionary origin is uncertain, the homology around the N terminus may provide a structural marker for a group of cytokines active on the pluripotential hemopoietic stem cell and its derivatives. PMID:3085095

  20. Orientation Dependence in Homologous Recombination

    PubMed Central

    Yamamoto, K.; Takahashi, N.; Fujitani, Y.; Yoshikura, H.; Kobayashi, I.

    1996-01-01

    Homologous recombination was investigated in Escherichia coli with two plasmids, each carrying the homologous region (two defective neo genes, one with an amino-end deletion and the other with a carboxyl-end deletion) in either direct or inverted orientation. Recombination efficiency was measured in recBC sbcBC and recBC sbcA strains in three ways. First, we measured the frequency of cells carrying neo(+) recombinant plasmids in stationary phase. Recombination between direct repeats was much more frequent than between inverted repeats in the recBC sbcBC strain but was equally frequent in the two substrates in the recBC sbcA strain. Second, the fluctuation test was used to exclude bias by a rate difference between the recombinant and parental plasmids and led to the same conclusion. Third, direct selection for recombinants just after transformation with or without substrate double-strand breaks yielded essentially the same results. Double-strand breaks elevated recombination in both the strains and in both substrates. These results are consistant with our previous findings that the major route of recombination in recBC sbcBC strains generates only one recombinant DNA from two DNAs and in recBC sbcA strains generates two recombinant DNAs from two DNAs. PMID:8722759

  1. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy

    PubMed Central

    Dai, Hanren; Wang, Yao; Lu, Xuechun

    2016-01-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy. PMID:26819347

  2. Humanized chimeric mouse models of hepatitis B virus infection.

    PubMed

    Sun, Suwan; Li, Jun

    2017-06-01

    Hepatitis B virus (HBV) infection is associated with an increased risk of hepatic cirrhosis, hepatocellular carcinoma, fulminant hepatitis and end-stage hepatic failure. Despite the availability of anti-HBV therapies, HBV infection remains a major global public health problem. Developing an ideal animal model of HBV infection to clarify the details of the HBV replication process, the viral life cycle, the resulting immunoresponse and the precise pathogenesis of HBV is difficult because HBV has an extremely narrow host range and almost exclusively infects humans. In this review, we summarize and evaluate animal models available for studying HBV infection, especially focusing on humanized chimeric mouse models, and we discuss future development trends regarding immunocompetent humanized mouse models that can delineate the natural history and immunopathophysiology of HBV infection. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  3. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy.

    PubMed

    Dai, Hanren; Wang, Yao; Lu, Xuechun; Han, Weidong

    2016-07-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy.

  4. The pharmacology of second-generation chimeric antigen receptors.

    PubMed

    van der Stegen, Sjoukje J C; Hamieh, Mohamad; Sadelain, Michel

    2015-07-01

    Second-generation chimeric antigen receptors (CARs) retarget and reprogramme T cells to augment their antitumour efficacy. The combined activating and co-stimulatory domains incorporated in these CARs critically determine the function, differentiation, metabolism and persistence of engineered T cells. CD19-targeted CARs that incorporate CD28 or 4-1BB signalling domains are the best known to date. Both have shown remarkable complete remission rates in patients with refractory B cell malignancies. Recent data indicate that CD28-based CARs direct a brisk proliferative response and boost effector functions, whereas 4-1BB-based CARs induce a more progressive T cell accumulation that may compensate for less immediate potency. These distinct kinetic features can be exploited to further develop CAR-based T cell therapies for a variety of cancers. A new field of immunopharmacology is emerging.

  5. Chimeric Antigen Receptor T Cell Therapy in Hematology.

    PubMed

    Ataca, Pınar; Arslan, Önder

    2015-12-01

    It is well demonstrated that the immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation. Adoptive T cell transfer has been improved to be more specific and potent and to cause less off-target toxicity. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR) and chimeric antigen receptor (CAR) modified T cells. On 1 July 2014, the United States Food and Drug Administration granted 'breakthrough therapy' designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the benefits of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical and clinical studies, and the effectiveness and drawbacks of this strategy.

  6. Chimeric antigen receptor T-cell therapy for ALL.

    PubMed

    Maude, Shannon L; Shpall, Elizabeth J; Grupp, Stephan A

    2014-12-05

    Relapsed and refractory leukemias pose substantial challenges in both children and adults, with very little progress being made in more than a decade. Targeted immunotherapy using chimeric antigen receptor (CAR)-modified T cells has emerged as a potent therapy with an innovative mechanism. Dramatic clinical responses with complete remission rates as high as 90% have been reported using CAR-modified T cells directed against the B-cell-specific antigen CD19 in patients with relapsed/refractory acute lymphoblastic leukemia. Supraphysiologic T-cell proliferation, a hallmark of this therapy, contributes to both efficacy and the most notable toxicity, cytokine release syndrome, posing a unique challenge for toxicity management. Further studies are necessary to identify additional targets, standardize approaches to cytokine release syndrome management, and determine the durability of remissions. © 2014 by The American Society of Hematology. All rights reserved.

  7. How Chimeric Antigen Receptor Design Affects Adoptive T Cell Therapy.

    PubMed

    Gacerez, Albert T; Arellano, Benjamine; Sentman, Charles L

    2016-12-01

    Chimeric antigen receptor (CAR) T cells have been developed to treat tumors and have shown great success against B cell malignancies. Exploiting modular designs and swappable domains, CARs can target an array of cell surface antigens and, upon receptor-ligand interactions, direct signaling cascades, thereby driving T cell effector functions. CARs have been designed using receptors, ligands, or scFv binding domains. Different regions of a CAR have each been found to play a role in determining the overall efficacy of CAR T cells. Therefore, this review provides an overview of CAR construction and common designs. Each CAR region is discussed in the context of its importance to a CAR's function. Additionally, the review explores how various engineering strategies have been applied to CAR T cells in order to regulate CAR T cell function and activity. J. Cell. Physiol. 231: 2590-2598, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. A Phenomenological and Dynamic View of Homology: Homologs as Persistently Reproducible Modules.

    PubMed

    Suzuki, Daichi G; Tanaka, Senji

    2017-01-01

    Homology is a fundamental concept in biology. However, the metaphysical status of homology, especially whether a homolog is a part of an individual or a member of a natural kind, is still a matter of intense debate. The proponents of the individuality view of homology criticize the natural kind view of homology by pointing out that homologs are subject to evolutionary transformation, and natural kinds do not change in the evolutionary process. Conversely, some proponents of the natural kind view of homology argue that a homolog can be construed both as a part of an individual and a member of a natural kind. They adopt the Homeostatic Property Cluster (HPC) theory of natural kinds, and the theory seems to strongly support their construal. Note that this construal implies the acceptance of essentialism. However, looking back on the history of the concept of homology, we should not overlook the fact that the individuality view was proposed to reject the essentialist interpretation of homology. Moreover, the essentialist notions of natural kinds can, in our view, mislead biologists about the phenomena of homology. Consequently, we need a non-essentialist view of homology, which we name the "persistently reproducible module" (PRM) view. This view highlights both the individual-like and kind-like aspects of homologs while stripping down both essentialist and anti-essentialist interpretations of homology. In this article, we articulate the PRM view of homology and explain why it is recommended over the other two views.

  9. Modeling cognition and disease using human glial chimeric mice.

    PubMed

    Goldman, Steven A; Nedergaard, Maiken; Windrem, Martha S

    2015-08-01

    As new methods for producing and isolating human glial progenitor cells (hGPCs) have been developed, the disorders of myelin have become especially compelling targets for cell-based therapy. Yet as animal modeling of glial progenitor cell-based therapies has progressed, it has become clear that transplanted hGPCs not only engraft and expand within murine hosts, but dynamically outcompete the resident progenitors so as to ultimately dominate the host brain. The engrafted human progenitor cells proceed to generate parenchymal astrocytes, and when faced with a hypomyelinated environment, oligodendrocytes as well. As a result, the recipient brains may become inexorably humanized with regards to their resident glial populations, yielding human glial chimeric mouse brains. These brains provide us a fundamentally new tool by which to assess the species-specific attributes of glia in modulating human cognition and information processing. In addition, the cellular humanization of these brains permits their use in studying glial infectious and inflammatory disorders unique to humans, and the effects of those disorders on the glial contributions to cognition. Perhaps most intriguingly, by pairing our ability to construct human glial chimeras with the production of patient-specific hGPCs derived from pluripotential stem cells, we may now establish mice in which a substantial proportion of resident glia are both human and disease-derived. These mice in particular may provide us new opportunities for studying the human-specific contributions of glia to psychopathology, as well as to higher cognition. As such, the assessment of human glial chimeric mice may provide us new insight into the species-specific contributions of glia to human cognitive evolution, as well as to the pathogenesis of human neurological and neuropsychiatric disease.

  10. Chimeric elk/mouse prion proteins in transgenic mice.

    PubMed

    Tamgüney, Gültekin; Giles, Kurt; Oehler, Abby; Johnson, Natrina L; DeArmond, Stephen J; Prusiner, Stanley B

    2013-02-01

    Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). Between codons 169 and 219, six residues distinguish ElkPrP from MoPrP: N169S, T173N, V183I, I202V, I214V and R219K. Using chimeric elk/mouse PrP constructs, we generated 12 Tg mouse lines and determined incubation times after intracerebral inoculation with the mouse-passaged RML scrapie or Elk1P CWD prions. Unexpectedly, one Tg mouse line expressing Elk3M(SNIVVK) exhibited incubation times of <70 days when inoculated with RML prions; a second line had incubation times of <90 days. In contrast, mice expressing full-length ElkPrP had incubation periods of >250 days for RML prions. Tg(Elk3M,SNIVVK) mice were less susceptible to CWD prions than Tg(ElkPrP) mice. Changing three C-terminal mouse residues (202, 214 and 219) to those of elk doubled the incubation time for mouse RML prions and rendered the mice resistant to Elk1P CWD prions. Mutating an additional two residues from mouse to elk at codons 169 and 173 increased the incubation times for mouse prions to >300 days, but made the mice susceptible to CWD prions. Our findings highlight the role of C-terminal residues in PrP that control the susceptibility and replication of prions.

  11. Chimeric conundra: are nucleomorphs and chromists monophyletic or polyphyletic?

    PubMed Central

    Cavalier-Smith, T; Allsopp, M T; Chao, E E

    1994-01-01

    All algae with chloroplasts located not freely in the cytosol, but inside two extra membranes, probably arose chimerically by the permanent fusion of two different eukaryote cells: a protozoan host and a eukaryotic algal symbiont. Two such groups, cryptomonads (phylum Cryptista) and Chlorarachniophyta, still retain a DNA-containing relic of the nucleus of the algal endosymbiont, known as the nucleomorph, as well as the host nucleus. These two phyla were traditionally assumed to have obtained their chloroplasts separately by two independent symbioses. We have sequenced the nuclear and the nucleomorph 18S rRNA genes of the nonphotosynthetic cryptomonad Chilomonas paramecium. Our phylogenetic analysis suggests that cryptomonad and chlorarachniophyte nucleomorphs may be related to each other and raises the possibility that both phyla may have diverged from a common ancestral chimeric cell that originated by a single endosymbiosis involving an algal endosymbiont related to the ancestor of red algae. But, because of the instability of the molecular trees when different taxa are added, there is insufficient evidence to overturn the traditional view that Chlorarachnion nucleomorphs evolved separately from a relative of green algae. The four phyla that contain chromophyte algae (those with chlorophyll c--i.e., Cryptista, Heterokonta, Haptophyta, Dinozoa) are distantly related to each other and to Chlorarachniophyta on our trees. However, all of the photosynthetic taxa within each of these four phyla radiate from each other very substantially after the radiation of the four phyla themselves. This favors the view that the common ancestor of these four phyla was not photosynthetic and that chloroplasts were implanted separately into each much more recently. This probable polyphyly of the chromophyte algae, if confirmed, would make it desirable to treat Cryptista, Heterokonta, and Haptophyta as separate kingdoms, rather than to group them together in the single kingdom

  12. Chimeric elk/mouse prion proteins in transgenic mice

    PubMed Central

    Tamgüney, Gültekin; Giles, Kurt; Oehler, Abby; Johnson, Natrina L.; DeArmond, Stephen J.

    2013-01-01

    Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). Between codons 169 and 219, six residues distinguish ElkPrP from MoPrP: N169S, T173N, V183I, I202V, I214V and R219K. Using chimeric elk/mouse PrP constructs, we generated 12 Tg mouse lines and determined incubation times after intracerebral inoculation with the mouse-passaged RML scrapie or Elk1P CWD prions. Unexpectedly, one Tg mouse line expressing Elk3M(SNIVVK) exhibited incubation times of <70 days when inoculated with RML prions; a second line had incubation times of <90 days. In contrast, mice expressing full-length ElkPrP had incubation periods of >250 days for RML prions. Tg(Elk3M,SNIVVK) mice were less susceptible to CWD prions than Tg(ElkPrP) mice. Changing three C-terminal mouse residues (202, 214 and 219) to those of elk doubled the incubation time for mouse RML prions and rendered the mice resistant to Elk1P CWD prions. Mutating an additional two residues from mouse to elk at codons 169 and 173 increased the incubation times for mouse prions to >300 days, but made the mice susceptible to CWD prions. Our findings highlight the role of C-terminal residues in PrP that control the susceptibility and replication of prions. PMID:23100369

  13. Novel fusion genes and chimeric transcripts in ependymal tumors.

    PubMed

    Olsen, Thale Kristin; Panagopoulos, Ioannis; Gorunova, Ludmila; Micci, Francesca; Andersen, Kristin; Kilen Andersen, Hege; Meling, Torstein R; Due-Tønnessen, Bernt; Scheie, David; Heim, Sverre; Brandal, Petter

    2016-12-01

    We have previously identified two ALK rearrangements in a subset of ependymal tumors using a combination of cytogenetic data and RNA sequencing. The aim of this study was to perform an unbiased search for fusion transcripts in our entire series of ependymal tumors. Fusion analysis was performed using the FusionCatcher algorithm on 12 RNA-sequenced ependymal tumors. Candidate transcripts were prioritized based on the software's filtering and manual visualization using the BLAST (Basic Local Alignment Search Tool) and BLAT (BLAST-like alignment tool) tools. Genomic and reverse transcriptase PCR with subsequent Sanger sequencing was used to validate the potential fusions. Fluorescent in situ hybridization (FISH) using locus-specific probes was also performed. A total of 841 candidate chimeric transcripts were identified in the 12 tumors, with an average of 49 unique candidate fusions per tumor. After algorithmic and manual filtering, the final list consisted of 24 potential fusion events. Raw RNA-seq read sequences and PCR validation supports two novel fusion genes: a reciprocal fusion gene involving UQCR10 and C1orf194 in an adult spinal ependymoma and a TSPAN4-CD151 fusion gene in a pediatric infratentorial anaplastic ependymoma. Our previously reported ALK rearrangements and the RELA and YAP1 fusions found in supratentorial ependymomas were until now the only known fusion genes present in ependymal tumors. The chimeric transcripts presented here are the first to be reported in infratentorial or spinal ependymomas. Further studies are required to characterize the genomic rearrangements causing these fusion genes, as well as the frequency and functional importance of the fusions. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. Pooled assembly of marine metagenomic datasets: enriching annotation through chimerism.

    PubMed

    Magasin, Jonathan D; Gerloff, Dietlind L

    2015-02-01

    Despite advances in high-throughput sequencing, marine metagenomic samples remain largely opaque. A typical sample contains billions of microbial organisms from thousands of genomes and quadrillions of DNA base pairs. Its derived metagenomic dataset underrepresents this complexity by orders of magnitude because of the sparseness and shortness of sequencing reads. Read shortness and sequencing errors pose a major challenge to accurate species and functional annotation. This includes distinguishing known from novel species. Often the majority of reads cannot be annotated and thus cannot help our interpretation of the sample. Here, we demonstrate quantitatively how careful assembly of marine metagenomic reads within, but also across, datasets can alleviate this problem. For 10 simulated datasets, each with species complexity modeled on a real counterpart, chimerism remained within the same species for most contigs (97%). For 42 real pyrosequencing ('454') datasets, assembly increased the proportion of annotated reads, and even more so when datasets were pooled, by on average 1.6% (max 6.6%) for species, 9.0% (max 28.7%) for Pfam protein domains and 9.4% (max 22.9%) for PANTHER gene families. Our results outline exciting prospects for data sharing in the metagenomics community. While chimeric sequences should be avoided in other areas of metagenomics (e.g. biodiversity analyses), conservative pooled assembly is advantageous for annotation specificity and sensitivity. Intriguingly, our experiment also found potential prospects for (low-cost) discovery of new species in 'old' data. dgerloff@ffame.org Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  15. Functional analysis of aldehyde oxidase using expressed chimeric enzyme between monkey and rat.

    PubMed

    Itoh, Kunio; Asakawa, Tasuku; Hoshino, Kouichi; Adachi, Mayuko; Fukiya, Kensuke; Watanabe, Nobuaki; Tanaka, Yorihisa

    2009-01-01

    Aldehyde oxidase (AO) is a homodimer with a subunit molecular mass of approximately 150 kDa. Each subunit consists of about 20 kDa 2Fe-2S cluster domain storing reducing equivalents, about 40 kDa flavine adenine dinucleotide (FAD) domain and about 85 kDa molybdenum cofactor (MoCo) domain containing a substrate binding site. In order to clarify the properties of each domain, especially substrate binding domain, chimeric cDNAs were constructed by mutual exchange of 2Fe-2S/FAD and MoCo domains between monkey and rat. Chimeric monkey/rat AO was referred to one with monkey type 2Fe-2S/FAD domains and a rat type MoCo domain. Rat/monkey AO was vice versa. AO-catalyzed 2-oxidation activities of (S)-RS-8359 were measured using the expressed enzyme in Escherichia coli. Substrate inhibition was seen in rat AO and chimeric monkey/rat AO, but not in monkey AO and chimeric rat/monkey AO, suggesting that the phenomenon might be dependent on the natures of MoCo domain of rat. A biphasic Eadie-Hofstee profile was observed in monkey AO and chimeric rat/monkey AO, but not rat AO and chimeric monkey/rat AO, indicating that the biphasic profile might be related to the properties of MoCo domain of monkey. Two-fold greater V(max) values were observed in monkey AO than in chimeric rat/monkey AO, and in chimeric monkey/rat AO than in rat AO, suggesting that monkey has the more effective electron transfer system than rat. Thus, the use of chimeric enzymes revealed that 2Fe-2S/FAD and MoCo domains affect the velocity and the quantitative profiles of AO-catalyzed (S)-RS-8359 2-oxidation, respectively.

  16. Generating chimeric mice from embryonic stem cells via vial coculturing or hypertonic microinjection.

    PubMed

    Lee, Kun-Hsiung

    2014-01-01

    The generation of a fertile embryonic stem cell (ESC)-derived or F0 (100 % coat color chimerism) mice is the final criterion in proving that the ESC is truly pluripotent. Many methods have been developed to produce chimeric mice. To date, the most popular methods for generating chimeric embryos is well sandwich aggregation between zona pellucida (ZP) removed (denuded) 2.5-day post-coitum (dpc) embryos and ESC clumps, or direct microinjection of ESCs into the cavity (blastocoel) of 3.5-dpc blastocysts. However, due to systemic limitations and the disadvantages of conventional microinjection, aggregation, and coculturing, two novel methods (vial coculturing and hypertonic microinjection) were developed in recent years at my laboratory.Coculturing 2.5-dpc denuded embryos with ESCs in 1.7-mL vials for ~3 h generates chimeras that have significantly high levels of chimerism (including 100 % coat color chimerism) and germline transmission. This method has significantly fewer instrumental and technological limitations than existing methods, and is an efficient, simple, inexpensive, and reproducible method for "mass production" of chimeric embryos. For laboratories without a microinjection system, this is the method of choice for generating chimeric embryos. Microinjecting ESCs into a subzonal space of 2.5-dpc embryos can generate germline-transmitted chimeras including 100 % coat color chimerism. However, this method is adopted rarely due to the very small and tight space between ZP and blastomeres. Using a laser pulse or Piezo-driven instrument/device to help introduce ESCs into the subzonal space of 2.5-dpc embryos demonstrates the superior efficiency in generating ESC-derived (F0) chimeras. Unfortunately, due to the need for an expensive instrument/device and extra fine skill, not many studies have used either method. Recently, ESCs injected into the large subzonal space of 2.5-dpc embryos in an injection medium containing 0.2-0.3 M sucrose very efficiently generated

  17. Cooperation of Cytokine Signaling with Chimeric Transcription Factors in Leukemogenesis: PML-Retinoic Acid Receptor Alpha Blocks Growth Factor-Mediated Differentiation

    PubMed Central

    Phan, Vernon T.; Shultz, David B.; Truong, Bao-Tran H.; Blake, Timothy J.; Brown, Anna L.; Gonda, Thomas J.; Beau, Michelle M. Le; Kogan, Scott C.

    2003-01-01

    We utilized a mouse model of acute promyelocytic leukemia (APL) to investigate how aberrant activation of cytokine signaling pathways interacts with chimeric transcription factors to generate acute myeloid leukemia. Expression in mice of the APL-associated fusion, PML-RARA, initially has only modest effects on myelopoiesis. Whereas treatment of control animals with interleukin-3 (IL-3) resulted in expanded myelopoiesis without a block in differentiation, PML-RARA abrogated differentiation that normally characterizes the response to IL-3. Retroviral transduction of bone marrow with an IL-3-expressing retrovirus revealed that IL-3 and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) combined to generate a lethal leukemia-like syndrome in <21 days. We also observed that a constitutively activated mutant IL-3 receptor, βcV449E, cooperated with PML-RARα in leukemogenesis, whereas a different activated mutant, βcI374N, did not. Analysis of additional mutations introduced into βcV449E showed that, although tyrosine phosphorylation of βc is necessary for cooperation, the Src homology 2 domain-containing transforming protein binding site is dispensable. Our results indicate that chimeric transcription factors can block the differentiative effects of growth factors. This combination can be potently leukemogenic, but the particular manner in which these types of mutations interact determines the ability of such combinations to generate acute myeloid leukemia. PMID:12808098

  18. Efficient signal transduction by a chimeric yeast-mammalian G protein alpha subunit Gpa1-Gsalpha covalently fused to the yeast receptor Ste2.

    PubMed Central

    Medici, R; Bianchi, E; Di Segni, G; Tocchini-Valentini, G P

    1997-01-01

    Saccharomyces cerevisiae uses G protein-coupled receptors for signal transduction. We show that a fusion protein between the alpha-factor receptor (Ste2) and the Galpha subunit (Gpa1) transduces the signal efficiently in yeast cells devoid of the endogeneous STE2 and GPA1 genes. To evaluate the function of different domains of Galpha, a chimera between the N-terminal region of yeast Gpa1 and the C-terminal region of rat Gsalpha has been constructed. This chimeric Gpa1-Gsalpha is capable of restoring viability to haploid gpa1Delta cells, but signal transduction is prevented. This is consistent with evidence showing that the C-terminus of the homologous Galpha is required for receptor-G protein recognition. Surprisingly, a fusion protein between Ste2 and Gpa1-Gsalpha is able to transduce the signal efficiently. It appears, therefore, that the C-terminus of Galpha is mainly responsible for bringing the G protein into the close proximity of the receptor's intracellular domains, thus ensuring efficient coupling, rather than having a particular role in transmitting the signal. To confirm this conclusion, we show that two proteins interacting with each other (such as Snf1 and Snf4, or Ras and Raf), each of them fused either to the receptor or to the chimeric Galpha, allow efficient signal transduction. PMID:9405353

  19. Khovanov homology of links and graphs

    NASA Astrophysics Data System (ADS)

    Stosic, Marko

    2006-05-01

    In this thesis we work with Khovanov homology of links and its generalizations, as well as with the homology of graphs. Khovanov homology of links consists of graded chain complexes which are link invariants, up to chain homotopy, with graded Euler characteristic equal to the Jones polynomial of the link. Hence, it can be regarded as the "categorification" of the Jones polynomial. We prove that the first homology group of positive braid knots is trivial. Futhermore, we prove that non-alternating torus knots are homologically thick. In addition, we show that we can decrease the number of full twists of torus knots without changing low-degree homology and consequently that there exists stable homology for torus knots. We also prove most of the above properties for Khovanov-Rozansky homology. Concerning graph homology, we categorify the dichromatic (and consequently Tutte) polynomial for graphs, by categorifying an infinite set of its one-variable specializations. We categorify explicitly the one-variable specialization that is an analog of the Jones polynomial of an alternating link corresponding to the initial graph. Also, we categorify explicitly the whole two-variable dichromatic polynomial of graphs by using Koszul complexes. textbf{Key-words:} Khovanov homology, Jones polynomial, link, torus knot, graph, dichromatic polynomial

  20. Homologous recombination and its regulation

    PubMed Central

    Krejci, Lumir; Altmannova, Veronika; Spirek, Mario; Zhao, Xiaolan

    2012-01-01

    Homologous recombination (HR) is critical both for repairing DNA lesions in mitosis and for chromosomal pairing and exchange during meiosis. However, some forms of HR can also lead to undesirable DNA rearrangements. Multiple regulatory mechanisms have evolved to ensure that HR takes place at the right time, place and manner. Several of these impinge on the control of Rad51 nucleofilaments that play a central role in HR. Some factors promote the formation of these structures while others lead to their disassembly or the use of alternative repair pathways. In this article, we review these mechanisms in both mitotic and meiotic environments and in different eukaryotic taxa, with an emphasis on yeast and mammal systems. Since mutations in several proteins that regulate Rad51 nucleofilaments are associated with cancer and cancer-prone syndromes, we discuss how understanding their functions can lead to the development of better tools for cancer diagnosis and therapy. PMID:22467216

  1. Interspecies homology of nitrogenase genes.

    PubMed Central

    Ruvkun, G B; Ausubel, F M

    1980-01-01

    Cloned nitrogen fixation (nif) genes from Klebsiella pneumoniae hybridize to DNA from 19 out of 19 widely divergent nitrogen-fixing bacterial strains but do not hybridize to DNA from 10 different non-nitrogen-fixing species. K. pneumoniae nif DNA fragments that hybridize to DNA from other species contain part of the three structural genes that code for nitrogenase polypeptides. We have utilized this homology to clone an EcoRI restriction endonuclease fragment from Rhizobium meliloti that hybridizes to the K. pneumoniae nif structural genes. Some of the species whose DNA hybridizes with K. pneumoniae nif DNA have been postulated to have diverged from K. pneumoniae 3 x 10(9) years ago. Nitrogenase genes are the only known example of such highly conserved prokaryotic translated genes. Nitrogenase genes are either extraordinarily conserved in evolution or have been exchanged between different nitrogen-fixing species relatively recently in evolutionary time. Images PMID:6987649

  2. Homologous artificial insemination and oligospermia.

    PubMed

    Speichinger, J P; Mattox, J H

    1976-02-01

    Of approximately 339 patients evaluated at a private infertility service over a 5-year period, 24 couples underwent homologous artificial insemination (AIH). Nineteen of these were performed to circumvent the problem of oligospermia, and only one pregancy was achieved in this group; conceivably this pregnancy could have occurred by chance. The difficulty in controlling the numerous variables in a clinical fertility study and the limitations of the present methodology are also discussed. It would appear that the use of AIH to circumvent oligospermia has not been successful. However, the present series is rather small. AIH should continue to be offered to couples who have a well-defined indication such as impotence, premature ejaculation, or any anatomical defect which prevents successful intromission. The use of AIH for patients with mild oligospermia but excellent sperm motility probably deserves a limited trial, since it is less expensive and may offer some chance for success.

  3. Homology and the hierarchy of biological systems.

    PubMed

    Sommer, Ralf J

    2008-07-01

    Homology is the similarity between organisms due to common ancestry. Introduced by Richard Owen in 1843 in a paper entitled "Lectures on comparative anatomy and physiology of the invertebrate animals", the concept of homology predates Darwin's "Origin of Species" and has been very influential throughout the history of evolutionary biology. Although homology is the central concept of all comparative biology and provides a logical basis for it, the definition of the term and the criteria of its application remain controversial. Here, I will discuss homology in the context of the hierarchy of biological organization. I will provide insights gained from an exemplary case study in evolutionary developmental biology that indicates the uncoupling of homology at different levels of biological organization. I argue that continuity and hierarchy are separate but equally important issues of homology. (c) 2008 Wiley Periodicals, Inc.

  4. Persistent homology analysis of craze formation

    NASA Astrophysics Data System (ADS)

    Ichinomiya, Takashi; Obayashi, Ippei; Hiraoka, Yasuaki

    2017-01-01

    We apply a persistent homology analysis to investigate the behavior of nanovoids during the crazing process of glassy polymers. We carry out a coarse-grained molecular dynamics simulation of the uniaxial deformation of an amorphous polymer and analyze the results with persistent homology. Persistent homology reveals the void coalescence during craze formation, and the results suggest that the yielding process is regarded as the percolation of nanovoids created by deformation.

  5. ChAy/Bx, a novel chimeric high-molecular-weight glutenin subunit gene apparently created by homoeologous recombination in Triticum turgidum ssp. dicoccoides.

    PubMed

    Guo, Xiao-Hui; Bi, Zhe-Guang; Wu, Bi-Hua; Wang, Zhen-Zhen; Hu, Ji-Liang; Zheng, You-Liang; Liu, Deng-Cai

    2013-12-01

    High-molecular-weight glutenin subunits (HMW-GSs) are of considerable interest, because they play a crucial role in determining dough viscoelastic properties and end-use quality of wheat flour. In this paper, ChAy/Bx, a novel chimeric HMW-GS gene from Triticum turgidum ssp. dicoccoides (AABB, 2n=4x=28) accession D129, was isolated and characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the electrophoretic mobility of the glutenin subunit encoded by ChAy/Bx was slightly faster than that of 1Dy12. The complete ORF of ChAy/Bx contained 1,671 bp encoding a deduced polypeptide of 555 amino acid residues (or 534 amino acid residues for the mature protein), making it the smallest HMW-GS gene known from Triticum species. Sequence analysis showed that ChAy/Bx was neither a conventional x-type nor a conventional y-type subunit gene, but a novel chimeric gene. Its first 1305 nt sequence was highly homologous with the corresponding sequence of 1Ay type genes, while its final 366 nt sequence was highly homologous with the corresponding sequence of 1Bx type genes. The mature ChAy/Bx protein consisted of the N-terminus of 1Ay type subunit (the first 414 amino acid residues) and the C-terminus of 1Bx type subunit (the final 120 amino acid residues). Secondary structure prediction showed that ChAy/Bx contained some domains of 1Ay subunit and some domains of 1Bx subunit. The special structure of this HMW glutenin chimera ChAy/Bx subunit might have unique effects on the end-use quality of wheat flour. Here we propose that homoeologous recombination might be a novel pathway for allelic variation or molecular evolution of HMW-GSs.

  6. Chimeric antigen receptor T-cell neuropsychiatric toxicity in acute lymphoblastic leukemia.

    PubMed

    Prudent, Vasthie; Breitbart, William S

    2017-01-04

    Chimeric antigen receptor T cells are used in the treatment of B-cell leukemias. Common chimeric antigen receptor T-cell toxicities can range from mild flu-like symptoms, such as fever and myalgia, to a more striking neuropsychiatric toxicity that can present as discrete neurological symptoms and delirium. We report here two cases of chimeric antigen receptor T-cell neuropsychiatric toxicity, one who presented as a mild delirium and aphasia that resolved without intervention, and one who presented with delirium, seizures, and respiratory insufficiency requiring intensive treatment. The current literature on the treatment and proposed mechanisms of this clinically challenging chimeric antigen receptor T-cell complication is also presented.

  7. Tolerance of Lung Allografts Achieved in Nonhuman Primates via Mixed Hematopoietic Chimerism

    PubMed Central

    Tonsho, M.; Lee, S.; Aoyama, A.; Boskovic, S.; Nadazdin, O.; Capetta, K.; Smith, R.-N.; Colvin, R. B.; Sachs, D. H.; Cosimi, A. B.; Kawai, T.; Madsen, J. C.; Benichou, G.; Allan, J. S.

    2015-01-01

    While the induction of transient mixed chimerism has tolerized MHC-mismatched renal grafts in nonhuman primates and patients, this approach has not been successful for more immunogenic organs. Here, we describe a modified delayed-tolerance-induction protocol resulting in three out of four monkeys achieving long-term lung allograft survival without ongoing immunosuppression. Two of the tolerant monkeys displayed stable mixed lymphoid chimerism, and the other showed transient chimerism. Serial biopsies and post-mortem specimens from the tolerant monkeys revealed no signs of chronic rejection. The tolerant recipients also exhibited T cell unresponsiveness and a lack of alloantibody. This is the first report of durable mixed chimerism and successful tolerance induction of MHC-mismatched lungs in primates. PMID:25904524

  8. A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells

    SciTech Connect

    Mourez, Thomas; Mesel-Lemoine, Mariana; Combredet, Chantal; Najburg, Valerie; Cayet, Nadege; Tangy, Frederic

    2011-10-25

    We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimeric particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes.

  9. Antiproliferative and GH-inhibitory activity of chimeric peptides consisting of GHRP-6 and somatostatin.

    PubMed

    Dasgupta, P; Singh, A T; Mukherjee, R

    1999-06-07

    Chimeric peptides consisting of growth hormone releasing peptide (GHRP-6) linked to somatostatin (6-11) via an amide bond to provide the effector parts of both the peptides were synthesized. The anti-proliferative, cytotoxic, and GH-inhibitory activities of these chimeric peptides were determined in vitro in the rat pituitary adenoma cell line GH3. One of the chimeric peptides, GSD, exhibited significantly greater (p < 0.001) anti-neoplastic and GH-inhibitory activity, as compared to RC-160. The hybrid peptides displayed high affinity binding to somatostatin receptors on GH3 cells. The bioactivity of GSD was found to be mediated by the stimulation of tyrosine phosphatase, involving a cGMP-dependent pathway, through pertussis toxin-sensitive G-proteins. Such potent GH-inhibitory chimeric peptides may be of potential importance in the therapy of acromegaly, as well as provide novel tools to study the regulation of GH secretion by GHRP and somatostatin.

  10. Tentative Mapping of Transcription-Induced Interchromosomal Interaction using Chimeric EST and mRNA Data

    PubMed Central

    Unneberg, Per; Claverie, Jean-Michel

    2007-01-01

    Recent studies on chromosome conformation show that chromosomes colocalize in the nucleus, bringing together active genes in transcription factories. This spatial proximity of actively transcribing genes could provide a means for RNA interaction at the transcript level. We have screened public databases for chimeric EST and mRNA sequences with the intent of mapping transcription-induced interchromosomal interactions. We suggest that chimeric transcripts may be the result of close encounters of active genes, either as functional products or “noise” in the transcription process, and that they could be used as probes for chromosome interactions. We have found a total of 5,614 chimeric ESTs and 587 chimeric mRNAs that meet our selection criteria. Due to their higher quality, the mRNA findings are of particular interest and we hope that they may serve as food for thought for specialists in diverse areas of molecular biology. PMID:17330142

  11. Identification of the rate of chimerism of different tissues with microsatellite markers in chicken chimeras.

    PubMed

    Siwek, Maria; Sławińska, Anna; Łakota, Paweł; Grajewski, Bartosz; Wawrzyńska, Magdalena; Wiśniewska, Ewa; Pławski, Andrzej; Słomski, Ryszard; Bednarczyk, Marek

    2010-01-01

    The goal of our study was to evaluate whether private alleles can be defined in microsatellite markers for the breeds under investigation; to evaluate if these private alleles distinguish chicken chimera when using different tissues; to trace them back to the donor: Green-Legged Partridgelike and recipient: White Leghorn chicken breeds, and further on, to estimate the level of chimerism in each tissue. Private and common alleles were defined for donor and recipient chicken breeds in 3 loci. The rate of chimerism was defined based on private alleles present in liver, heart, breast muscle, femoral muscle and gonads. The highest rate of chimerism was observed in liver. A lower rate of chimersim was observed in gonads, and femoral muscle, and finally the lowest rate of chimerism was observed in breast muscle and heart.

  12. Homology-independent metrics for comparative genomics.

    PubMed

    Coutinho, Tarcisio José Domingos; Franco, Glória Regina; Lobo, Francisco Pereira

    2015-01-01

    A mainstream procedure to analyze the wealth of genomic data available nowadays is the detection of homologous regions shared across genomes, followed by the extraction of biological information from the patterns of conservation and variation observed in such regions. Although of pivotal importance, comparative genomic procedures that rely on homology inference are obviously not applicable if no homologous regions are detectable. This fact excludes a considerable portion of "genomic dark matter" with no significant similarity - and, consequently, no inferred homology to any other known sequence - from several downstream comparative genomic methods. In this review we compile several sequence metrics that do not rely on homology inference and can be used to compare nucleotide sequences and extract biologically meaningful information from them. These metrics comprise several compositional parameters calculated from sequence data alone, such as GC content, dinucleotide odds ratio, and several codon bias metrics. They also share other interesting properties, such as pervasiveness (patterns persist on smaller scales) and phylogenetic signal. We also cite examples where these homology-independent metrics have been successfully applied to support several bioinformatics challenges, such as taxonomic classification of biological sequences without homology inference. They where also used to detect higher-order patterns of interactions in biological systems, ranging from detecting coevolutionary trends between the genomes of viruses and their hosts to characterization of gene pools of entire microbial communities. We argue that, if correctly understood and applied, homology-independent metrics can add important layers of biological information in comparative genomic studies without prior homology inference.

  13. Frequency of chimerism in populations of the kelp Lessonia spicata in central Chile

    PubMed Central

    2017-01-01

    Chimerism occurs when two genetically distinct conspecific individuals fuse together generating a single entity. Coalescence and chimerism in red seaweeds has been positively related to an increase in body size, and the consequent reduction in susceptibility to mortality factors, thus increasing survival, reproductive potential and tolerance to stress in contrast to genetically homogeneous organisms. In addition, they showed that a particular pattern of post-fusion growth maintains higher genetic diversity and chimerism in the holdfast but homogenous axes. In Chilean kelps (brown seaweeds), intraorganismal genetic heterogeneity (IGH) and holdfast coalescence has been described in previous research, but the extent of chimerism in wild populations and the patterns of distribution of the genetically heterogeneous thallus zone have scarcely been studied. Since kelps are under continuous harvesting, with enormous social, ecological and economic importance, natural chimerism can be considered a priceless in-situ reservoir of natural genetic resources and variability. In this study, we therefore examined the frequency of IGH and chimerism in three harvested populations of Lessonia spicata. We then evaluated whether chimeric wild-type holdfasts show higher genetic diversity than erect axes (stipe and lamina) and explored the impact of this on the traditional estimation of genetic diversity at the population level. We found a high frequency of IGH (60–100%) and chimerism (33.3–86.7%), varying according to the studied population. We evidenced that chimerism occurs mostly in holdfasts, exhibiting heterogeneous tissues, whereas stipes and lamina were more homogeneous, generating a vertical gradient of allele and genotype abundance as well as divergence, constituting the first time “within- plant” genetic patterns have been reported in kelps. This is very different from the chimeric patterns described in land plants and animals. Finally, we evidenced that IGH affected

  14. Frequency of chimerism in populations of the kelp Lessonia spicata in central Chile.

    PubMed

    González, Alejandra V; Santelices, Bernabé

    2017-01-01

    Chimerism occurs when two genetically distinct conspecific individuals fuse together generating a single entity. Coalescence and chimerism in red seaweeds has been positively related to an increase in body size, and the consequent reduction in susceptibility to mortality factors, thus increasing survival, reproductive potential and tolerance to stress in contrast to genetically homogeneous organisms. In addition, they showed that a particular pattern of post-fusion growth maintains higher genetic diversity and chimerism in the holdfast but homogenous axes. In Chilean kelps (brown seaweeds), intraorganismal genetic heterogeneity (IGH) and holdfast coalescence has been described in previous research, but the extent of chimerism in wild populations and the patterns of distribution of the genetically heterogeneous thallus zone have scarcely been studied. Since kelps are under continuous harvesting, with enormous social, ecological and economic importance, natural chimerism can be considered a priceless in-situ reservoir of natural genetic resources and variability. In this study, we therefore examined the frequency of IGH and chimerism in three harvested populations of Lessonia spicata. We then evaluated whether chimeric wild-type holdfasts show higher genetic diversity than erect axes (stipe and lamina) and explored the impact of this on the traditional estimation of genetic diversity at the population level. We found a high frequency of IGH (60-100%) and chimerism (33.3-86.7%), varying according to the studied population. We evidenced that chimerism occurs mostly in holdfasts, exhibiting heterogeneous tissues, whereas stipes and lamina were more homogeneous, generating a vertical gradient of allele and genotype abundance as well as divergence, constituting the first time "within- plant" genetic patterns have been reported in kelps. This is very different from the chimeric patterns described in land plants and animals. Finally, we evidenced that IGH affected genetic

  15. Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency

    PubMed Central

    Yin, HaiFang; Boisguerin, Prisca; Moulton, Hong M; Betts, Corinne; Seow, Yiqi; Boutilier, Jordan; Wang, Qingsong; Walsh, Anthony; Lebleu, Bernard; Wood, Matthew JA

    2013-01-01

    We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies. PMID:24064708

  16. Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency.

    PubMed

    Yin, Haifang; Boisguerin, Prisca; Moulton, Hong M; Betts, Corinne; Seow, Yiqi; Boutilier, Jordan; Wang, Qingsong; Walsh, Anthony; Lebleu, Bernard; Wood, Matthew Ja

    2013-09-24

    We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.Molecular Therapy-Nucleic Acids (2013) 2, e124; doi:10.1038/mtna.2013

  17. Mixed donor chimerism in non-malignant haematological diseases after allogeneic bone marrow transplantation.

    PubMed

    Shamshad, Ghassan Umair; Ahmed, Suhaib; Bhatti, Farhat Abbas; Ali, Nadir

    2012-12-01

    To determine the frequency of mixed donor chimerism in patients of non-malignant haematological diseases after allogeneic bone marrow transplant. A cross-sectional, observational study. Department of Haematology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from July 2010 to June 2011. Donor chimerism was assessed in patients of aplastic anaemia and beta-thalassaemia major who underwent allogeneic bone marrow transplantation (BMT). Peripheral blood samples were used to assess chimerism status by analysis of short tandem repeats (STR). In patients where pre-transplant blood sample was not available, swab of buccal mucosa was used for pre-transplant STR profile. A standard set of primers for STR markers were used and the amplified DNA was resolved by gel electrophoresis and stained with silver nitrate. The percentage of donor origin DNA was estimated by densitometer. Out of 84 patients, 52 (62%) were males, while 32 (38%) were females. In patients of beta-thalassaemia major, 31 (62%) developed mixed donor chimerism (MC), 13 (26%) developed complete donor chimerism (CC) and 6 (12%) had graft failure. In aplastic anaemia, 17 patients (50%) achieved MC, 13 (38.2%) had CC and 4 (11.8%) developed graft failure. The combined frequency of mixed donor chimerism for both the diseases was 58.3%. D3S1358 was the most informative STR marker in these patients. Majority of the studied patients developed mixed donor chimerism following bone marrow transplantation, whereas only a minor percentage of the patients had graft failure. Analysis of D3S1358 was the most informative in assessing donor chimerism in patients who underwent BMT.

  18. Chimeric cDNA Sequences from Citrus tristeza virus Confer RNA Silencing-Mediated Resistance in Transgenic Nicotiana benthamiana Plants.

    PubMed

    Roy, Gourgopal; Sudarshana, Mysore R; Ullman, Diane E; Ding, Shou-Wei; Dandekar, Abhaya M; Falk, Bryce W

    2006-08-01

    ABSTRACT RNA silencing has been shown to be an important mechanism for conferring resistance in transgenic, virus-resistant plants. We used this approach to evaluate resistance in Nicotiana benthamiana plants transformed with chimeric coding and noncoding sequences from Citrus tristeza virus (CTV). Several independent transgenic plant lines were generated, using two constructs (pCTV1 and pCTV2) designed to produce self-complementary transcripts. The pCTV1 contained cDNA sequences from the CTV capsid protein (CP), p20, and 3' untranslated region (UTR); and pCTV2 contained CP, p23, and 3' UTR sequences. Heterologous recombinant Potato virus X (PVX) containing either homologous or heterologous CTV sequences was used to challenge plants and resistance was evaluated phenotypically and validated with reverse-transcriptase polymerase chain reaction and northern hybridization analysis. Transgenic plants (T1 generation) for each construct showed resistance to recombinant PVX constructs used for challenge experiments when PVX contained p20 or UTR (for CTV1 plants), or p23 or UTR (for CTV2 plants). However, no resistance was seen when plants were challenged with PVX containing the CTV CP. T2 generation plants also showed resistance even when challenged with PVX containing the cognate CTV sequences obtained from heterologous CTV isolates. The presence of transgene-specific small interfering RNAs in the resistant CTV1 and CTV2 plants indicated that resistance was mediated by post-transcriptional gene silencing.

  19. Chimerism reveals a role for the streptokinase Beta -domain in nonproteolytic active site formation, substrate, and inhibitor interactions.

    PubMed

    Gladysheva, Inna P; Sazonova, Irina Y; Chowdhry, Shakeel A; Liu, Lin; Turner, Ryan B; Reed, Guy L

    2002-07-26

    Streptokinase (SK) and staphylokinase form cofactor-enzyme complexes that promote the degradation of fibrin thrombi by activating human plasminogen. The unique abilities of streptokinase to nonproteolytically activate plasminogen or to alter the interactions of plasmin with substrates and inhibitors may be the result of high affinity binding mediated by the streptokinase beta-domain. To examine this hypothesis, a chimeric streptokinase, SKbetaswap, was created by swapping the SK beta-domain with the homologous beta-domain of Streptococcus uberis Pg activator (SUPA or PauA, SK uberis), a streptokinase that cannot activate human plasminogen. SKbetaswap formed a tight complex with microplasminogen with an affinity comparable with streptokinase. The SKbetaswap-plasmin complex also activated human plasminogen with catalytic efficiencies (k(cat)/K(m) = 16.8 versus 15.2 microm(-1) min(-1)) comparable with streptokinase. However, SKbetaswap was incapable of nonproteolytic active site generation and activated plasminogen by a staphylokinase mechanism. When compared with streptokinase complexes, SKbetaswap-plasmin and SKbetaswap-microplasmin complexes had altered affinities for low molecular weight substrates. The SKbetaswap-plasmin complex also was less resistant than the streptokinase-plasmin complex to inhibition by alpha(2)-antiplasmin and was readily inhibited by soybean trypsin inhibitor. Thus, in addition to mediating high affinity binding to plasmin(ogen), the streptokinase beta-domain is required for nonproteolytic active site generation and specifically modulates the interactions of the complex with substrates and inhibitors.

  20. Regulated expression of the feline panleukopenia virus P38 promoter on extrachromosomal FPV/EBV chimeric plasmids.

    PubMed Central

    Clemens, D L; Carlson, J O

    1989-01-01

    Feline panleukopenia virus/Epstein-Barr virus (FPV/EBV) chimeric expression plasmids were constructed to study regulation of the structural protein gene of the parvovirus, FPV, in a homologous cell culture system. Detection and quantitation of activity from the native FPV promoter, P38, was facilitated by fusing the Escherichia coli lacZ gene with the FPV structural protein gene. Feline cell lines which stably maintained these plasmids extrachromosomally were established. Constitutive beta-galactosidase activity was low but increased up to 40-fold after infection with FPV. Expression of beta-galactosidase was only detected when the FPV/lacZ gene was oriented in the same transcriptional direction as the Epstein-Barr virus gene coding for EBNA-1. When a small open reading frame upstream of the FPV/lacZ initiation codon was deleted, beta-galactosidase expression increased another 4.7- to 26-fold. These changes in beta-galactosidase activity indicate that expression of the FPV structural protein gene is regulated both transcriptionally and posttranscriptionally. Images PMID:2542586

  1. Novel noncoding RNA from human Y distal heterochromatic block (Yq12) generates testis-specific chimeric CDC2L2

    PubMed Central

    Jehan, Zeenath; Vallinayagam, Sambandam; Tiwari, Shrish; Pradhan, Suman; Singh, Lalji; Suresh, Amritha; Reddy, Hemakumar M.; Ahuja, Y.R.; Jesudasan, Rachel A.

    2007-01-01

    The human Y chromosome, because it is enriched in repetitive DNA, has been very intractable to genetic and molecular analyses. There is no previous evidence for developmental stage- and testis-specific transcription from the male-specific region of the Y (MSY). Here, we present evidence for the first time for a developmental stage- and testis-specific transcription from MSY distal heterochromatic block. We isolated two novel RNAs, which localize to Yq12 in multiple copies, show testis-specific expression, and lack active X-homologs. Experimental evidence shows that one of the above Yq12 noncoding RNAs (ncRNAs) trans-splices with CDC2L2 mRNA from chromosome 1p36.3 locus to generate a testis-specific chimeric β sv13 isoform. This 67-nt 5′UTR provided by the Yq12 transcript contains within it a Y box protein-binding CCAAT motif, indicating translational regulation of the β sv13 isoform in testis. This is also the first report of trans-splicing between a Y chromosomal and an autosomal transcript. PMID:17095710

  2. Chimeric Reverse Temporal Muscle and Pericranial Flap for Double-Layer Closure of Deep Facial Defects.

    PubMed

    Algan, Said; Tan, Onder; Kara, Murat; Inaloz, Akin; Cakmak, Mehmet Akif; Aydin, Osman Enver

    2017-05-24

    Volumetric or multiplane defects of the upper and midface remain a challenge for reconstruction because of limited regional flap options. In this study, the authors harvested the reverse temporalis muscle flap and pericranial flap (RTMP flap) based on the same vascular pedicle, the superficial temporal artery, in a chimeric manner to obtain double-layer closure of deep facial defects. This study was a prospective case series performed in the Department of Plastic Surgery of Ataturk University (Erzurum, Turkey). The outcomes, including flap survival, postoperative complications, reconstructive success, esthetic appearance, and donor site morbidity, were clinically evaluated. Fourteen patients (10 male and 4 female) with deep defects of the middle third of the face underwent reconstruction using the chimeric RTMP flap. All chimeric RTMP flaps survived without postoperative complications. All defects were successfully repaired and covered with chimeric RTMP flaps. Patients were satisfied with the esthetic results. The chimeric RTMP flap is a good reconstruction option and can be used safely for moderate to large 3-dimensional defects of the middle and upper face. Smooth and durable coverage over the bulky muscle flap used to fill the volume defect and a larger flap for larger volume defects can be obtained by including the pericranial segment of the chimeric RTMP flap. Copyright © 2017 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

  3. Chimerism and cure: hematologic and pathologic correction of murine sickle cell disease.

    PubMed

    Kean, Leslie S; Manci, Elizabeth A; Perry, Jennifer; Balkan, Can; Coley, Shana; Holtzclaw, David; Adams, Andrew B; Larsen, Christian P; Hsu, Lewis L; Archer, David R

    2003-12-15

    Bone marrow transplantation (BMT) is the only curative therapy for sickle cell disease (SCD). However, the morbidity and mortality related to pretransplantation myeloablative chemotherapy often outweighs the morbidity of SCD itself, thus severely limiting the number of patients eligible for transplantation. Although nonmyeloablative transplantation is expected to reduce the risk of BMT, it will likely result in mixed-chimerism rather than complete replacement with donor stem cells. Clinical application of nonmyeloablative transplantation thus requires knowledge of the effect of mixed chimerism on SCD pathophysiology. We have, therefore, created a panel of transplanted SCD mice that received transplants displaying an array of red blood cell (RBC) and white blood cell (WBC) chimerism. A significant enrichment of RBC over WBC chimerism occurred in these mice, because of the dramatic survival advantage of donor over sickle RBCs in the peripheral blood. Increasing levels of RBC chimerism provided progressive correction of hematologic and pathologic abnormalities. However, sickle bone marrow and splenic hematopoiesis was not corrected until peripheral blood sickle RBCs were fully replaced with donor RBCs. These results have important and unexpected implications for nonmyeloablative BMT for SCD. As the critical hematopoietic organs were not corrected without full RBC replacement, 100% peripheral blood RBC chimerism becomes the most important benchmark for cure after nonmyeloablative BMT.

  4. Tetraploid cells of enhanced green fluorescent protein transgenic mice in tetraploid/diploid-chimeric embryos.

    PubMed

    Ishiguro, Naomi; Kano, Kiyoshi; Yamamoto, Yoshio; Taniguchi, Kazuyuki

    2005-10-01

    We succeeded in noninvasively analyzing the distribution of tetraploid (4n) cells in tetraploid<-->diploid (4n<-->2n) chimeric embryos by using enhanced green fluorescent protein (EGFP) transgenic (Tg) mouse embryos. We also evaluated whether this technique of analyzing 4n-cells in EGFP Tg 4n<-->2n chimeric embryos could be used to determine which characteristics of 4n-cells cause the death of 4n-embryos and restricted distribution of 4n-cells in 4n<-->2n-chimeric embryos after implantation. In our experiments, the distribution of 4n-cells in 4n<-->2n-embryos was normal until an embryonic age of 3.5 days (E3.5). With respect to morphological development, there were no differences between 4n-, diploid (2n), 4n<-->2n-, and diploid/diploid (2n<-->2n) chimeric embryos, but the number of cells in the tetraploid (4n) blastocyst was smaller than expected. This decrease in the number of cells may have caused cell death or reduced the rate of cell division in 4n-cells, and may have restricted the distribution of 4n-cells in 4n<-->2n-chimeric embryos. This study demonstrated the utility of EGFP transgenic mouse embryos for relatively easy and noninvasive study of the sequential distribution of cells in chimeric embryos.

  5. Antistaphylococcal activity of bacteriophage derived chimeric protein P128.

    PubMed

    Vipra, Aradhana A; Desai, Srividya Narayanamurthy; Roy, Panchali; Patil, Raghu; Raj, Juliet Mohan; Narasimhaswamy, Nagalakshmi; Paul, Vivek Daniel; Chikkamadaiah, Ravisha; Sriram, Bharathi

    2012-03-22

    Bacterial drug resistance is one of the most significant challenges to human health today. In particular, effective antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA) are urgently needed. A causal relationship between nasal commensal S. aureus and infection has been reported. Accordingly, elimination of nasal S. aureus reduces the risk of infection. Enzymes that degrade bacterial cell walls show promise as antibacterial agents. Bacteriophage-encoded bacterial cell wall-degrading enzymes exhibit intrinsic bactericidal activity. P128 is a chimeric protein that combines the lethal activity of the phage tail-associated muralytic enzyme of Phage K and the staphylococcal cell wall targeting-domain (SH3b) of lysostaphin.Here we report results of in vitro studies evaluating the susceptibility of staphylococcal strains to this novel protein. Using the broth microdilution method adapted for lysostaphin, we found that P128 is effective against S. aureus clinical strains including MRSA, methicillin-sensitive S. aureus (MSSA), and a mupirocin-resistant S. aureus. Minimum bactericidal concentrations and minimum inhibitory concentrations of P128 (1-64 μg/mL) were similar across the 32 S. aureus strains tested, demonstrating its bactericidal nature.In time-kill assays, P128 reduced colony-forming units by 99.99% within 1 h and inhibited growth up to 24 h.In an assay simulating topical application of P128 to skin or other biological surfaces, P128 hydrogel was efficacious when layered on cells seeded on solid media. P128 hydrogel was lethal to Staphylococci recovered from nares of healthy people and treated without any processing or culturing steps, indicating its in situ efficacy. This methodology used for in vitro assessment of P128 as an agent for eradicating nasal carriage is unique. The novel chimeric protein P128 is a staphylococcal cell wall-degrading enzyme under development for clearance of S. aureus nasal colonization and MRSA infection. The

  6. Equivalence of Heegaard Floer homology and embedded contact homology via open book decompositions

    PubMed Central

    Colin, Vincent; Ghiggini, Paolo; Honda, Ko

    2011-01-01

    We sketch the proof of the equivalence between the hat versions of Heegaard Floer homology and embedded contact homology (ECH). The key point is to express these two Floer homology theories in terms of an open book decomposition of the ambient manifold. PMID:21525415

  7. Buoyancy instability of homologous implosions

    NASA Astrophysics Data System (ADS)

    Johnson, Bryan

    2015-11-01

    Hot spot turbulence is a potential contributor to yield degradation in inertial confinement fusion (ICF) capsules, although its origin, if present, remains unclear. In this work, a perturbation analysis is performed of an analytical homologous solution that mimics the hot spot and surrounding cold fuel during the late stages of an ICF implosion. It is shown that the flow is governed by the Schwarzschild criterion for buoyant stability, and that during stagnation, short wavelength entropy and vorticity fluctuations amplify by a factor exp (π |N0 | ts) , where N0 is the buoyancy frequency at stagnation and ts is the stagnation time scale. This amplification factor is exponentially sensitive to mean flow gradients and varies from 103-107 for realistic gradients. Comparisons are made with a Lagrangian hydrodynamics code, and it is found that a numerical resolution of ~ 30 zones per wavelength is required to capture the evolution of vorticity accurately. This translates to an angular resolution of ~(12 / l) ∘ , or ~ 0 .1° to resolve the fastest growing modes (Legendre mode l > 100).

  8. [Symmetries and homologies of Geomerida].

    PubMed

    Zarenkov, N A

    2005-01-01

    The symmetry of Earths life cover (Geomerida) was described generally by L.A. Zenkevich (1948). It coincides with the symmetry of geographic cover. Its symmetry elements are equatorial plane and three meridonal planes corresponded to oceans and continents. The hypsographic curve with point of inflection (symmetry element) on 3 km depth line should be added to these elements. The plankton and benthos communities as well as fauna of taxons are distributed symmetrically according these symmetry elements. Zenkevich model was successfully extrapolated to plankton by K.V. Beklemishev (1967, 1969) and to abyssal benthos by Sokolova M.N. (1986). The plankton communities inhabiting symmetrically located macrocirculations are considered as homologous. The character of circulation determines the trophic structure of plankton and benthos. In the case of high productivity of plankton, benthic grazing animals feed on sedimented particles have bilateral symmetric mouthpart. Otherwise they have to acquire food from water column and use cyclomeric mouthpart. Thus, the symmetry of macrocirculations determines the symmetry distribution of benthic animals with two major symmetries of mouthparts. The peculiarities of organisms' symmetry are discussed in the context of Pierre Curie principle and the ideas of K.V. Beklemishev concerning evolution of morphological axes.

  9. Homology in classical and molecular biology.

    PubMed

    Patterson, C

    1988-11-01

    Hypotheses of homology are the basis of comparative morphology and comparative molecular biology. The kinds of homologous and nonhomologous relations in classical and molecular biology are explored through the three tests that may be applied to a hypothesis of homology: congruence, conjunction, and similarity. The same three tests apply in molecular comparisons and in morphology, and in each field they differentiate eight kinds of relation. These various relations are discussed and compared. The unit or standard of comparison differs in morphology and in molecular biology; in morphology it is the adult or life cycle, but with molecules it is the haploid genome. In morphology the congruence test is decisive in separating homology and nonhomology, whereas with molecular sequence data similarity is the decisive test. Consequences of this difference are that the boundary between homology and nonhomology is not the same in molecular biology as in morphology, that homology and synapomorphy can be equated in morphology but not in all molecular comparisons, and that there is no detected molecular equivalent of convergence. Since molecular homology may reflect either species phylogeny or gene phylogeny, there are more kinds of homologous relation between molecular sequences than in morphology. The terms paraxenology and plerology are proposed for two of these kinds--respectively, the consequence of multiple xenology and of gene conversion.

  10. GENE SEQUENCE HOMOLOGY OF CHEMOKINES ACROSS SPECIES

    USDA-ARS?s Scientific Manuscript database

    The abundance of expressed gene and protein sequences available in the biological information databases facilitates comparison of protein homologies. A high degree of sequence similarity typically implies homology regarding structure and function and may provide clues to antibody cross-react...

  11. DNA Sequence Alignment during Homologous Recombination*

    PubMed Central

    Greene, Eric C.

    2016-01-01

    Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination. PMID:27129270

  12. DNA Sequence Alignment during Homologous Recombination.

    PubMed

    Greene, Eric C

    2016-05-27

    Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Random-walk model of homologous recombination

    NASA Astrophysics Data System (ADS)

    Fujitani, Youhei; Kobayashi, Ichizo

    1995-12-01

    Interaction between two homologous (i.e., identical or nearly identical) DNA sequences leads to their homologous recombination in the cell. We present the following stochastic model to explain the dependence of the frequency of homologous recombination on the length of the homologous region. The branch point connecting the two DNAs in a reaction intermediate follows the random-walk process along the homology (N base-pairs). If the branch point reaches either of the homology ends, it bounds back to the homologous region at a probability of γ (reflection coefficient) and is destroyed at a probability of 1-γ. When γ is small, the frequency of homologous recombination is found to be proportional to N3 for smaller N and a linear function of N for larger N. The exponent of the nonlinear dependence for smaller N decreases from three as γ increases. When γ=1, only the linear dependence is left. These theoretical results can explain many experimental data in various systems. (c) 1995 The American Physical Society

  14. Protective and immunological behavior of chimeric yellow fever dengue vaccine.

    PubMed

    Halstead, Scott B; Russell, Philip K

    2016-03-29

    Clinical observations from the third year of the Sanofi Pasteur chimeric yellow fever dengue tetravalent vaccine (CYD) trials document both protection and vaccination-enhanced dengue disease among vaccine recipients. Children who were 5 years-old or younger when vaccinated experienced a DENV disease resulting in hospitalization at 5 times the rate of controls. On closer inspection, hospitalized cases among vaccinated seropositives, those at highest risk to hospitalized disease accompanying a dengue virus (DENV) infection, were greatly reduced by vaccination. But, seronegative individuals of all ages after being vaccinated were only modestly protected from mild to moderate disease throughout the entire observation period despite developing neutralizing antibodies at high rates. Applying a simple epidemiological model to the data, vaccinated seronegative individuals of all ages were at increased risk of developing hospitalized disease during a subsequent wild type DENV infection. The etiology of disease in placebo and vaccinated children resulting in hospitalization during a DENV infection, while clinically similar are of different origin. The implications of the observed mixture of DENV protection and enhanced disease in CYD vaccinees are discussed.

  15. Competitive annealing of multiple DNA origami: formation of chimeric origami

    NASA Astrophysics Data System (ADS)

    Majikes, Jacob M.; Nash, Jessica A.; LaBean, Thomas H.

    2016-11-01

    Scaffolded DNA origami are a robust tool for building discrete nanoscale objects at high yield. This strategy ensures, in the design process, that the desired nanostructure is the minimum free energy state for the designed set of DNA sequences. Despite aiming for the minimum free energy structure, the folding process which leads to that conformation is difficult to characterize, although it has been the subject of much research. In order to shed light on the molecular folding pathways, this study intentionally frustrates the folding process of these systems by simultaneously annealing the staple pools for multiple target or parent origami structures, forcing competition. A surprising result of these competitive, simultaneous anneals is the formation of chimeric DNA origami which inherit structural regions from both parent origami. By comparing the regions inherited from the parent origami, relative stability of substructures were compared. This allowed examination of the folding process with typical characterization techniques and materials. Anneal curves were then used as a means to rapidly generate a phase diagram of anticipated behavior as a function of staple excess and parent staple ratio. This initial study shows that competitive anneals provide an exciting way to create diverse new nanostructures and may be used to examine the relative stability of various structural motifs.

  16. Chimeric antigen receptor T-cell therapies for lymphoma.

    PubMed

    Brudno, Jennifer N; Kochenderfer, James N

    2017-08-31

    New therapies are needed for patients with Hodgkin or non-Hodgkin lymphomas that are resistant to standard therapies. Indeed, unresponsiveness to standard chemotherapy and relapse after autologous stem-cell transplantation are indicators of an especially poor prognosis. Chimeric antigen receptor (CAR) T cells are emerging as a novel treatment modality for these patients. Clinical trial data have demonstrated the potent activity of anti-CD19 CAR T cells against multiple subtypes of B-cell lymphoma, including diffuse large-B-cell lymphoma (DLBCL), follicular lymphoma, mantle-cell lymphoma, and marginal-zone lymphoma. Importantly, anti-CD19 CAR T cells have impressive activity against chemotherapy-refractory lymphoma, inducing durable complete remissions lasting >2 years in some patients with refractory DLBCL. CAR-T-cell therapies are, however, associated with potentially fatal toxicities, including cytokine-release syndrome and neurological toxicities. CAR T cells with novel target antigens, including CD20, CD22, and κ-light chain for B-cell lymphomas, and CD30 for Hodgkin and T-cell lymphomas, are currently being investigated in clinical trials. Centrally manufactured CAR T cells are also being tested in industry-sponsored multicentre clinical trials, and will probably soon become a standard therapy. Herein, we review the clinical efficacy and toxicity of CAR-T-cell therapies for lymphoma, and discuss their limitations and future directions with regard to toxicity management, CAR designs and CAR-T-cell phenotypes, conditioning regimens, and combination therapies.

  17. Chimeric Antigen Receptor T Cells in Hematologic Malignancies.

    PubMed

    Shank, Brandon R; Do, Bryan; Sevin, Adrienne; Chen, Sheree E; Neelapu, Sattva S; Horowitz, Sandra B

    2017-03-01

    Patients with B-cell hematologic malignancies who progress through first- or second-line chemotherapy have a poor prognosis. Early clinical trials with autologous anti-CD19 chimeric antigen receptor (CAR) T cells have demonstrated promising results for patients who have relapsed or refractory disease. Lymphodepleting conditioning regimens, including cyclophosphamide, fludarabine, pentostatin, bendamustine, interleukin-2, and total body irradiation, are often administered before the infusion of CAR T cells, allowing for greater T-cell expansion. The major toxicity associated with CAR T-cell infusions is cytokine release syndrome (CRS), a potentially life-threatening systemic inflammatory disorder. The quick onset and progression of CRS require rapid detection and intervention to reduce treatment-related mortality. Management with tocilizumab can help ameliorate the symptoms of severe CRS, allowing steroids, which diminish the expansion and persistence of CAR T cells, to be reserved for tocilizumab-refractory patients. Other toxicities of CAR T-cell therapy include neutropenia and/or febrile neutropenia, infection, tumor lysis syndrome, neurotoxicity and nausea/vomiting. A review of patients' medications is imperative to eliminate medications that may contribute to treatment-related toxicities. Studies are ongoing to help optimize patient selection, preparation, safety, and management of individuals receiving CAR T cells. Long-term follow-up will help establish the place of CAR T cells in therapy.

  18. Chimeric influenza haemagglutinins: Generation and use in pseudotype neutralization assays.

    PubMed

    Ferrara, Francesca; Temperton, Nigel

    2017-01-01

    Recently chimeric influenza haemagglutinins (cHAs) have been generated as potential 'universal' vaccination antigens and as tools to identify HA stalk-directed antibodies via their use as antigens in ELISA, and virus or pseudotype-based neutralization assays. The original methods [1], [2] used for their generation require the amplification of regions of interest (head and stalk) using primers containing SapI sites and subsequent cloning into pDZ plasmid. This requires precise primer design, checking for the absence of SapI sites in the sequence of interest, and multi-segment ligation. As an alternative strategy we have developed and optimized a new protocol for assembling the cHA by exploiting Gibson Assembly. •This method also requires precise primer design, but it is rapid and methodologically simple to perform. We have evaluated that using this method it is possible to construct a cHA encoding DNA in less than a week.•Additional weeks are however necessary to optimize the production of pseudotyped lentiviral particles and to perform neutralization assays using them as surrogate antigens.•In comparison to the original protocols, we have also observed that performing parallel neutralization assays using pseudotypes harbouring the two parental HAs, permits effective delineation between stalk and head antibody responses in the samples tested.

  19. Chimeric antigen receptor-modified T cells strike back

    PubMed Central

    Frigault, Matthew J.

    2016-01-01

    Chimeric antigen receptors (CARs) are engineered molecules designed to endow a polyclonal T-cell population with the ability to recognize tumor-associated surface antigens. In their simplest form, CARs comprise a targeting moiety in the form of a single-chain variable fragment from an antibody connected to various intracellular signaling domains allowing for T-cell activation. This powerful approach combines the specificity of an antibody with the cytotoxic ability of a T cell. There has been much excitement since early phase trials of CAR-T cells targeting CD19 expressed on B-cell malignancies demonstrated remarkable efficacy in inducing long-term, stable remissions in otherwise relapsed/refractory disease. Despite these successes, we have just begun to understand the intricacies of CAR biology with efforts underway to utilize this platform in the treatment of other, previously refractory malignancies. Challenges currently include identification of viable cancer targets, management strategies for potentially severe and irreversible toxicities and overcoming the immunosuppressive nature of the tumor microenvironment. This review will focus on basic CAR structure and function, previous success and new approaches aimed at the broader application of CAR-T-cell therapy. PMID:27021308

  20. Engineering HIV-Specific Immunity with Chimeric Antigen Receptors.

    PubMed

    Kitchen, Scott G; Zack, Jerome A

    2016-12-01

    HIV remains a highly important public health and clinical issue despite many recent advances in attempting to develop a cure, which has remained elusive for most people infected with HIV. HIV disease can be controlled with pharmacologic therapies; however, these treatments are expensive, may have severe side effects, and are not curative. Consequently, an improved means to control or eliminate HIV replication is needed. Cytotoxic T lymphocytes (CTLs) play a critical role in controlling viral replication and are an important part in the ability of the immune response to eradicate most viral infections. There are considerable efforts to enhance CTL responses in HIV-infected individuals in hopes of providing the immune response with armaments to more effectively control viral replication. In this review, we discuss some of these efforts and focus on the development of a gene therapy-based approach to engineer hematopoietic stem cells with an HIV-1-specific chimeric antigen receptor, which seeks to provide an inexhaustible source of HIV-1-specific immune cells that are MHC unrestricted and superior to natural antiviral T cell responses. These efforts provide the basis for further development of T cell functional enhancement to target and treat chronic HIV infection in hopes of eradicating the virus from the body.

  1. Future directions in chimeric antigen receptor T cell therapy.

    PubMed

    Maude, Shannon L

    2017-02-01

    The impact of immunotherapy has grown exponentially in the past 5 years. Principle illustrations are encouraging results with engineered T cells expressing a chimeric antigen receptor (CAR). This experimental therapy is developing simultaneously in pediatric and adult clinical trials, making this field particularly relevant and exciting for pediatric oncologists. CAR-modified T cells targeting CD19 have produced dramatic antitumor responses in patients with relapsed/refractory B cell acute lymphoblastic leukemia. Clinical trials from several institutions, in both children and adults, using distinct CAR T cell products have demonstrated similar high complete remission rates of 61-93%, with durable remissions observed. Although the development of CARs for other malignancies has lagged behind, research into novel approaches to overcome inherent challenges is promising. Clinical trials of CAR-modified T cells have produced unprecedented results and are anticipated to have a broader impact as this approach expands into other indications, including other cancers and frontline therapy. The potential for long-term disease control, if fully realized, will have a transformative impact on the field.

  2. Current status of chimeric antigen receptor therapy for haematological malignancies.

    PubMed

    Maude, Shannon; Barrett, David M

    2016-01-01

    The field of adoptive cell transfer includes chimeric antigen receptor (CAR) engineered T cells, constructs that emerged from basic research into principles of immunology and have transformed into clinically effective therapies for haematological malignancies. T cells engineered to express these artificial receptors hold great promise, but also carry significant risk. While permanent genetic modification of mature T cells appears safe, modulating their in vivo function is difficult, partly because the robust response can trigger other arms of the immune system. Suicide systems and toxicity management with cytokine blockade or signal transduction modulators have emerged as a new frontier in this field, a far cry from early problems getting CAR T cells to work at all. Currently, clinical trials in patients with relapsed or refractory B cell malignancies treated with CD19-specific CAR T cells have induced durable remissions in adults and children. Results from these trials indicate that more work needs to be done to understand biomarkers of efficacy, the role of T cell persistence and how to integrate this care into standard practice. Cell therapy will not be a 'one size fits all' class of medicine, and here we will discuss the development of this therapy and important questions for its future. © 2015 John Wiley & Sons Ltd.

  3. Long-term assessment of particulate matter using CHIMERE model

    NASA Astrophysics Data System (ADS)

    Monteiro, A.; Miranda, A. I.; Borrego, C.; Vautard, R.; Ferreira, J.; Perez, A. T.

    Particulate matter (PM) and aerosols have became a critical pollutant and object of several research applications, due to their increasing levels, especially in urban areas, causing air pollution problems and thus effects on human health. The main purpose of this study is to perform a first long-term air quality assessment for Portugal, regarding aerosols and PM pollution. The CHIMERE chemistry-transport model, forced by the MM5 meteorological fields, was applied over Portugal for 2001 year, with 10 km horizontal resolution, using an emission inventory obtained from a spatial top-down disaggregation of the 2001 national inventory database. The evaluation model exercise shows a model trend to overestimate particulate pollution episodes (peaks) at urban sites, especially in winter season. This could be due to an underprediction of the winter model vertical mixing and also to an overestimation of PM emissions. Simulated inorganic components (ammonium and sulfate) and secondary organic aerosols (SOA) were compared to measurements taken at Aveiro (northwest coast of Portugal). An underestimation of the three components was verified. However, the model is able to predict their seasonal variation. Nevertheless, as a first approach, and despite the complex topography and coastal location of Portugal affected by sea salt natural aerosols emissions, the results obtained show that the model reproduces the PM levels, temporal evolution, and spatial patterns. The concentration maps reveal that the areas with high PM values are covered by the air quality monitoring network.

  4. Toxicities of chimeric antigen receptor T cells: recognition and management

    PubMed Central

    Brudno, Jennifer N.

    2016-01-01

    Chimeric antigen receptor (CAR) T cells can produce durable remissions in hematologic malignancies that are not responsive to standard therapies. Yet the use of CAR T cells is limited by potentially severe toxicities. Early case reports of unexpected organ damage and deaths following CAR T-cell therapy first highlighted the possible dangers of this new treatment. CAR T cells can potentially damage normal tissues by specifically targeting a tumor-associated antigen that is also expressed on those tissues. Cytokine release syndrome (CRS), a systemic inflammatory response caused by cytokines released by infused CAR T cells can lead to widespread reversible organ dysfunction. CRS is the most common type of toxicity caused by CAR T cells. Neurologic toxicity due to CAR T cells might in some cases have a different pathophysiology than CRS and requires different management. Aggressive supportive care is necessary for all patients experiencing CAR T-cell toxicities, with early intervention for hypotension and treatment of concurrent infections being essential. Interleukin-6 receptor blockade with tocilizumab remains the mainstay pharmacologic therapy for CRS, though indications for administration vary among centers. Corticosteroids should be reserved for neurologic toxicities and CRS not responsive to tocilizumab. Pharmacologic management is complicated by the risk of immunosuppressive therapy abrogating the antimalignancy activity of the CAR T cells. This review describes the toxicities caused by CAR T cells and reviews the published approaches used to manage toxicities. We present guidelines for treating patients experiencing CRS and other adverse events following CAR T-cell therapy. PMID:27207799

  5. Chimeric protein repair of laminin polymerization ameliorates muscular dystrophy phenotype.

    PubMed

    McKee, Karen K; Crosson, Stephanie C; Meinen, Sarina; Reinhard, Judith R; Rüegg, Markus A; Yurchenco, Peter D

    2017-03-01

    Mutations in laminin α2-subunit (Lmα2, encoded by LAMA2) are linked to approximately 30% of congenital muscular dystrophy cases. Mice with a homozygous mutation in Lama2 (dy2J mice) express a nonpolymerizing form of laminin-211 (Lm211) and are a model for ambulatory-type Lmα2-deficient muscular dystrophy. Here, we developed transgenic dy2J mice with muscle-specific expression of αLNNd, a laminin/nidogen chimeric protein that provides a missing polymerization domain. Muscle-specific expression of αLNNd in dy2J mice resulted in strong amelioration of the dystrophic phenotype, manifested by the prevention of fibrosis and restoration of forelimb grip strength. αLNNd also restored myofiber shape, size, and numbers to control levels in dy2J mice. Laminin immunostaining and quantitation of tissue extractions revealed increased Lm211 expression in αLNNd-transgenic dy2J mice. In cultured myotubes, we determined that αLNNd expression increased myotube surface accumulation of polymerization-deficient recombinant laminins, with retention of collagen IV, reiterating the basement membrane (BM) changes observed in vivo. Laminin LN domain mutations linked to several of the Lmα2-deficient muscular dystrophies are predicted to compromise polymerization. The data herein support the hypothesis that engineered expression of αLNNd can overcome polymerization deficits to increase laminin, stabilize BM structure, and substantially ameliorate muscular dystrophy.

  6. AMKL chimeric transcription factors are potent inducers of leukemia.

    PubMed

    Dang, J; Nance, S; Ma, J; Cheng, J; Walsh, M P; Vogel, P; Easton, J; Song, G; Rusch, M; Gedman, A L; Koss, C; Downing, J R; Gruber, T A

    2017-03-10

    Acute megakaryoblastic leukemia in patients without Down syndrome is a rare malignancy with a poor prognosis. RNA sequencing of fourteen pediatric cases previously identified novel fusion transcripts that are predicted to be pathological including CBFA2T3-GLIS2, GATA2-HOXA9, MN1-FLI and NIPBL-HOXB9. In contrast to CBFA2T3-GLIS2, which is insufficient to induce leukemia, we demonstrate that the introduction of GATA2-HOXA9, MN1-FLI1 or NIPBL-HOXB9 into murine bone marrow induces overt disease in syngeneic transplant models. With the exception of MN1, full penetrance was not achieved through the introduction of fusion partner genes alone, suggesting that the chimeric transcripts possess a unique gain-of-function phenotype. Leukemias were found to exhibit elements of the megakaryocyte erythroid progenitor gene expression program, as well as unique leukemia-specific signatures that contribute to transformation. Comprehensive genomic analyses of resultant murine tumors revealed few cooperating mutations confirming the strength of the fusion genes and their role as pathological drivers. These models are critical for both the understanding of the biology of disease as well as providing a tool for the identification of effective therapeutic agents in preclinical studies.Leukemia advance online publication, 10 March 2017; doi:10.1038/leu.2017.51.

  7. Chimeric Antigen Receptor (CAR)-Engineered Lymphocytes for Cancer Therapy

    PubMed Central

    Ramos, Carlos A.; Dotti, Gianpietro

    2011-01-01

    Introduction Chimeric antigen receptors (CARs) usually combine the antigen binding site of a monoclonal antibody with the signal activating machinery of a T cell, freeing antigen recognition from major histocompatibility complex restriction and thus breaking one of the barriers to more widespread application of cellular therapy. Similar to treatment strategies employing monoclonal antibodies, T cells expressing CARs are highly targeted, but additionally offer the potential benefits of active trafficking to tumor sites, in vivo expansion and long term persistence. Furthermore, gene transfer allows the introduction of countermeasures to tumor immune evasion and of safety mechanisms. Areas covered The authors review the basic structure of so-called first and later generation CARs and their potential advantages over other immune therapy systems. It is described how these molecules can be grafted into immune cells (including retroviral and non-retroviral transduction methods) and strategies to improve the in vivo persistence and function of immune cells expressing CARs are discussed. Examples of tumor associated antigens that have been targeted in preclinical models are presented and clinical experience with these modified cells is summarized. Finally, a discussion on safety issues surrounding CAR gene transfer into T cells and potential solutions to them, are presented. Expert opinion Because of recent advances in immunology, genetics and cell processing, CAR-modified T cells will likely play an increasing role in the cellular therapy of cancer, chronic infections and autoimmune disorders. PMID:21463133

  8. Computational methods for remote homolog identification.

    PubMed

    Wan, Xiu-Feng; Xu, Dong

    2005-12-01

    As more and more protein sequences are available, homolog identification becomes increasingly important for functional, structural, and evolutional studies of proteins. Many homologous proteins were separated a very long time ago in their evolutionary history and thus their sequences share low sequence identity. These remote homologs have become a research focus in bioinformatics over the past decade, and some significant advances have been achieved. In this paper, we provide a comprehensive review on computational techniques used in remote homolog identification based on different methods, including sequence-sequence comparison, and sequence-structure comparison, and structure-structure comparison. Other miscellaneous approaches are also summarized. Pointers to the online resources of these methods and their related databases are provided. Comparisons among different methods in terms of their technical approaches, their strengths, and limitations are followed. Studies on proteins in SARS-CoV are shown as an example for remote homolog identification application.

  9. [Hydrolysis of chimeric proteins by enteropeptidase at the specific linker (Asp)4Lys depending on refolding conditions].

    PubMed

    Shibanova, E D; Mikhaĭlova, A G; Aleksandrov, S L; Rumsh, L D

    2000-07-01

    Refolding from inclusion bodies of chimeric proteins containing the enteropeptidase-specific linker (Asp)4Lys was carried out. It was shown that, depending on the refolding conditions, chimeric proteins function as substrates or inhibitors of the enteropeptidase. The efficiency of the enteropeptidase hydrolysis of chimeric proteins containing the (Asp)4Lys linker may depend not only on the amino acid sequence of the protein binding site for the enzyme but also on the site conformation.

  10. Engineering, expression and purification of a chimeric fibrin-specific streptokinase.

    PubMed

    Taheri, Mohammad Naser; Behzad-Behbahani, Abbas; Rafiei Dehbidi, Gholamreza; Salehi, Saeede; Sharifzadeh, Sedigheh

    2016-12-01

    Streptokinase is a valuable fibrinolytic agent used to cope with myocardial infarction and brain stroke. Despite its high efficiency in dissolving blood clots, streptokinase (SK) has no specificity in binding fibrin, causing some problems such as internal bleedings following its administration. To make streptokinase fibrin specific and limit the fibrinolytic process to the clot location, we engineered a chimeric streptokinase by fusing the fibrin binding Kringle 2 domain of tissue plasminogen activator (TPA) to the streptokinase N-terminal end. The chimeric SK construct (KSK) with inserted Kringle 2 domain was cloned into pET28a expression vector. The expression of recombinant protein was carried out in Escherichia coli origami (DE3) and confirmed by SDS-PAGE and Western blotting analyses. We used the chromogenic substrate S-2251 method to assess the specific activities of the chimeric and control wild-type proteins. Then, the two proteins were added in amounts with equal activity to fibrin clots of identical size. Finally, the supernatant above the fibrin clots was collected and subjected to the chromogenic assay to analyze the specificity of the chimeric protein. The specific activities of the chimeric and wild-type proteins were found to be 0.06 U/mg and 0.07 U/mg, respectively. Because of the binding of the chimeric protein to fibrin, the mean specific activity was significantly lower in the KSK supernatant (0.01) compared with the control (approximately 0.06) (p < 0.05). Our in vitro results indicate that the chimeric streptokinase protein has strong fibrin-specific activity compared to the wild-type protein. However, further in vivo studies are needed to evaluate its potential fibrinolytic effects. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Early Chimerism After Liver Transplantation Reflects the Clinical Course of Recurrent Hepatitis C.

    PubMed

    Utsumi, Masashi; Takaki, Akinobu; Umeda, Yuzo; Koike, Kazuko; Napier, Stephanie C; Watanabe, Nobukazu; Shinoura, Susumu; Yoshida, Ryuichi; Nobuoka, Daisuke; Yasunaka, Tetsuya; Oto, Takahiro; Araki, Motoo; Yamamoto, Kazuhide; Fujiwara, Toshiyoshi; Yagi, Takahito

    2017-03-24

    BACKGROUND Human leukocyte antigen (HLA) mismatch is a characteristic feature of post-orthotopic liver transplantation (OLT) hepatitis C. To investigate the importance of donor HLA-restricted immune cells in post-OLT hepatitis C recurrence, we analyzed the frequency of donor chimerism and the clinical course of post-OLT hepatitis C. MATERIAL AND METHODS We analyzed peripheral blood chimerism in 11 HCV-reinfected patients with post-HLA mismatched OLT. Patients were divided into 2 groups: the OLT chronic hepatitis C (CHC) group (n=8), exhibiting active hepatitis C recurrence; and the OLT-persistently normal ALT (PNALT) group (n=3), without active hepatitis. Chimerism was analyzed by flow cytometry using donor-specific anti-HLA antibodies in peripheral blood mononuclear cells from 1-100 days after OLT. Kidney (n=7) and lung (n=7) transplant recipients were also analyzed for comparison. As immune cells from the donor liver might contribute to post-OLT chimerism, the characteristics of perfusates from donor livers (n=10) were analyzed and defined. RESULTS Donor-derived cells were frequently observed in liver and lung transplant recipients. The frequency of donor-derived cells from the B cell subset was significantly higher in peripheral blood from OLT-CHC group than in that of the OLT-PNALT group. B cells, however, were not the predominant subset in the perfusates, indicating that inflow of donor-derived cells alone did not cause the chimerism. CONCLUSIONS Chimerism of B cells is frequent in liver transplant patients with early recurrence of hepatitis C. We propose that monitoring of early chimerism could facilitate early detection of chronic hepatitis C recurrence, although we need more cases to investigate.

  12. Preventing Elevated Radix Deformity in Asian Rhinoplasty with a Chimeric Dorsal-Glabellar Construct

    PubMed Central

    Zelken, Jonathan A.; Hong, Joon Pio; Broyles, Justin M.; Hsiao, Yen-Chang

    2016-01-01

    Background Asian facial aesthetic surgery should enhance, but not change, natural features. Augmentation rhinoplasty is a hallmark of Asian cosmetic surgery. In the authors' experience, I-shaped implants can elevate and efface the radix, leading to an unnatural appearance (elevated radix deformity). Objectives The Chimeric technique was developed to control final radix position and preserve the nasal profile. We aim to demonstrate that the Chimeric technique promotes forward projection, not elevation, of the radix. Methods Between 2013 and 2015, 49 patients underwent rhinoplasty with I-shaped implants. Nineteen patients had Chimeric dorsal-glabellar implants, 30 did not. Standardized photographs were obtained at every visit. Novel and established photogrammetric parameters were used to describe radix position and position change. A retrospective chart review provided additional procedural details and outcomes data. Results Patients were followed for 10.8 months (range, 2-36 months). Nasal height increase (113% vs 107%) and bridge length increase (118% vs 105%) were significantly greater when the Chimeric technique was not performed (P < .0001). The nasofrontal angle increased 6° in both groups; there was no difference between groups. The vector of radix position change was 26.1° in the Chimeric group and 63.4° in the traditional group (P < .0001). Conclusions The Chimeric technique preserves the nasal profile with a favorable (horizontal) radix transposition vector. There was not a significant difference in final radix position when Chimeric rhinoplasty was performed because that is controlled by implant thickness and position. The technique did not blunt the radix significantly. Level of Evidence: 4 Therapeutic PMID:26879296

  13. Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein

    SciTech Connect

    Ye Ling; Sun Yuliang; Lin Jianguo; Bu Zhigao; Wu Qingyang; Jiang, Shibo; Steinhauer, David A.; Compans, Richard W.; Yang Chinglai . E-mail: chyang@emory.edu

    2006-08-15

    The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV.

  14. Influence of conditioning regimens and stem cell sources on donor-type chimerism early after stem cell transplantation.

    PubMed

    Sugita, Junichi; Tanaka, Junji; Hashimoto, Aya; Shiratori, Souichi; Yasumoto, Atsushi; Wakasa, Kentaro; Kikuchi, Misato; Shigematsu, Akio; Miura, Yoko; Tsutsumi, Yutaka; Kondo, Takeshi; Asaka, Masahiro; Imamura, Masahiro

    2008-12-01

    We retrospectively analyzed very early chimerism before and ongoing neutrophil engraftment (days 7, 14, 21, 28) and investigated the influence of conditioning regimens and stem cell sources on donor-type chimerism in 59 Japanese patients who had received allogeneic hematopoietic stem cell transplantation. The percentage of donor-type chimerism increased before engraftment in all patients who achieved engraftment. The average percentage of donor-type chimerism in patients who had received reduced-intensity stem cell transplantation (RIST) with total body irradiation (TBI) was significantly higher than that in patients who had received RIST without TBI (98.8% vs 87.5% on day 21, P<0.01; 99.3% vs 84.3% on day 28, P<0.01). The average percentage of donor-type chimerism after peripheral blood stem cell transplantation was significantly higher than that after bone marrow transplantation on day 7 (81.5% vs 43.1%, P<0.01), and the average percentage of donor-type chimerism after cord blood transplantation was significantly lower on day 14 (55.8% vs 84.8%, P<0.05). Compared with the average percentage of donor-type chimerism in patients who achieved engraftment with each stem cell source, a notable decrease in donor-type chimerism was observed in patients who failed to achieve engraftment. This study suggests that differences in conditioning regimens and stem cell sources should be taken into account when considering donor-type chimerism.

  15. ChimerDB 3.0: an enhanced database for fusion genes from cancer transcriptome and literature data mining

    PubMed Central

    Lee, Myunggyo; Lee, Kyubum; Yu, Namhee; Jang, Insu; Choi, Ikjung; Kim, Pora; Jang, Ye Eun; Kim, Byounggun; Kim, Sunkyu; Lee, Byungwook; Kang, Jaewoo; Lee, Sanghyuk

    2017-01-01

    Fusion gene is an important class of therapeutic targets and prognostic markers in cancer. ChimerDB is a comprehensive database of fusion genes encompassing analysis of deep sequencing data and manual curations. In this update, the database coverage was enhanced considerably by adding two new modules of The Cancer Genome Atlas (TCGA) RNA-Seq analysis and PubMed abstract mining. ChimerDB 3.0 is composed of three modules of ChimerKB, ChimerPub and ChimerSeq. ChimerKB represents a knowledgebase including 1066 fusion genes with manual curation that were compiled from public resources of fusion genes with experimental evidences. ChimerPub includes 2767 fusion genes obtained from text mining of PubMed abstracts. ChimerSeq module is designed to archive the fusion candidates from deep sequencing data. Importantly, we have analyzed RNA-Seq data of the TCGA project covering 4569 patients in 23 cancer types using two reliable programs of FusionScan and TopHat-Fusion. The new user interface supports diverse search options and graphic representation of fusion gene structure. ChimerDB 3.0 is available at http://ercsb.ewha.ac.kr/fusiongene/. PMID:27899563

  16. ChimerDB 3.0: an enhanced database for fusion genes from cancer transcriptome and literature data mining.

    PubMed

    Lee, Myunggyo; Lee, Kyubum; Yu, Namhee; Jang, Insu; Choi, Ikjung; Kim, Pora; Jang, Ye Eun; Kim, Byounggun; Kim, Sunkyu; Lee, Byungwook; Kang, Jaewoo; Lee, Sanghyuk

    2017-01-04

    Fusion gene is an important class of therapeutic targets and prognostic markers in cancer. ChimerDB is a comprehensive database of fusion genes encompassing analysis of deep sequencing data and manual curations. In this update, the database coverage was enhanced considerably by adding two new modules of The Cancer Genome Atlas (TCGA) RNA-Seq analysis and PubMed abstract mining. ChimerDB 3.0 is composed of three modules of ChimerKB, ChimerPub and ChimerSeq. ChimerKB represents a knowledgebase including 1066 fusion genes with manual curation that were compiled from public resources of fusion genes with experimental evidences. ChimerPub includes 2767 fusion genes obtained from text mining of PubMed abstracts. ChimerSeq module is designed to archive the fusion candidates from deep sequencing data. Importantly, we have analyzed RNA-Seq data of the TCGA project covering 4569 patients in 23 cancer types using two reliable programs of FusionScan and TopHat-Fusion. The new user interface supports diverse search options and graphic representation of fusion gene structure. ChimerDB 3.0 is available at http://ercsb.ewha.ac.kr/fusiongene/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Rats and mice immunised with chimeric human/mouse proteinase 3 produce autoantibodies to mouse Pr3 and rat granulocytes

    PubMed Central

    van der Geld, Ymke M; Hellmark, Thomas; Selga, Daina; Heeringa, Peter; Huitema, Minke G; Limburg, Pieter C; Kallenberg, Cees G M

    2007-01-01

    Aim In this study, we employed chimeric human/mouse Proteinase 3 (PR3) proteins as tools to induce an autoantibody response to PR3 in rats and mice. Method Rats and mice were immunised with recombinant human PR3 (HPR3), recombinant murine PR3 (mPR3), single chimeric human/mouse PR3 (HHm, HmH, mHH, mmH, mHm, Hmm) or pools of chimeric proteins. Antibodies to mPR3 and HPR3 were measured by ELISA. Antibodies to rat PR3 were determined by indirect immunofluorescence (IIF) on rat white blood cells. Urinalysis was performed by dipstick analysis. Kidney and lung tissue was obtained for pathological examination. Results In mice, immunisation with the chimeric human/mouse PR3 Hmm led to an autoantibody response to mPR3. Rats immunised with the chimeric human/mouse PR3 Hmm, HmH and mmH, or a pool of the chimeric human/mouse PR3 proteins, produced antibodies selectively binding to rat granulocytes as detected by IIF. No gross pathological abnormalities could be detected in kidney or lungs of mice or rats immunised with chimeric human/mouse PR3. Conclusion Immunisation with chimeric human/mouse proteins induces autoantibodies to PR3 in rats and mice. Chimeric proteins can be instrumental in developing experimental models for autoimmune diseases. PMID:17644551

  18. Homology--history of a concept.

    PubMed

    Panchen, A L

    1999-01-01

    The concept of homology is traceable to Aristotle, but Belon's comparison in 1555 of a human skeleton with that of a bird expressed it overtly. Before the late 18th century, the dominant view of the pattern of organisms was the scala naturae--even Linnaeus with his divergent hierarchical classification did not necessarily see the resulting taxonomic pattern as a natural phenomenon. The divergent hierarchy, rather than the acceptance of phylogeny, was the necessary spur to discussion of homology and the concept of analogy. Lamarck, despite his proposal of evolution, attributed homology to his escalator naturae and analogy to convergent acquired characters. Significantly, it was the concept of serial homology that emerged at the end of the 18th century, although comparison between organisms became popular soon after, and was boosted by the famous Cuvier/Geoffroy Saint-Hilaire debate of the 1830s. The concepts of homology and analogy were well understood by the pre- (or anti-) evolutionary comparative anatomists before the general acceptance of phylogeny, and they were defined by Owen in 1843. The acceptance of evolution led to the idea that homology should be defined by common ancestry, and to the confusion between definition and explanation. The term 'homoplasy', introduced by Lankester in 1870, also arose from a phylogenetic explanation of homology.

  19. Duplication of chicken defensin7 gene generated by gene conversion and homologous recombination.

    PubMed

    Lee, Mi Ok; Bornelöv, Susanne; Andersson, Leif; Lamont, Susan J; Chen, Junfeng; Womack, James E

    2016-11-29

    Defensins constitute an evolutionary conserved family of cationic antimicrobial peptides that play a key role in host innate immune responses to infection. Defensin genes generally reside in complex genomic regions that are prone to structural variation, and defensin genes exhibit extensive copy number variation in humans and in other species. Copy number variation of defensin genes was examined in inbred lines of Leghorn and Fayoumi chickens, and a duplication of defensin7 was discovered in the Fayoumi breed. Analysis of junction sequences confirmed the occurrence of a simple tandem duplication of defensin7 with sequence identity at the junction, suggesting nonallelic homologous recombination between defensin7 and defensin6 The duplication event generated two chimeric promoters that are best explained by gene conversion followed by homologous recombination. Expression of defensin7 was not elevated in animals with two genes despite both genes being transcribed in the tissues examined. Computational prediction of promoter regions revealed the presence of several putative transcription factor binding sites generated by the duplication event. These data provide insight into the evolution and possible function of large gene families and specifically, the defensins.

  20. Duplication of chicken defensin7 gene generated by gene conversion and homologous recombination

    PubMed Central

    Lee, Mi Ok; Bornelöv, Susanne; Andersson, Leif; Lamont, Susan J.; Chen, Junfeng; Womack, James E.

    2016-01-01

    Defensins constitute an evolutionary conserved family of cationic antimicrobial peptides that play a key role in host innate immune responses to infection. Defensin genes generally reside in complex genomic regions that are prone to structural variation, and defensin genes exhibit extensive copy number variation in humans and in other species. Copy number variation of defensin genes was examined in inbred lines of Leghorn and Fayoumi chickens, and a duplication of defensin7 was discovered in the Fayoumi breed. Analysis of junction sequences confirmed the occurrence of a simple tandem duplication of defensin7 with sequence identity at the junction, suggesting nonallelic homologous recombination between defensin7 and defensin6. The duplication event generated two chimeric promoters that are best explained by gene conversion followed by homologous recombination. Expression of defensin7 was not elevated in animals with two genes despite both genes being transcribed in the tissues examined. Computational prediction of promoter regions revealed the presence of several putative transcription factor binding sites generated by the duplication event. These data provide insight into the evolution and possible function of large gene families and specifically, the defensins. PMID:27849592

  1. Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells.

    PubMed

    Stroncek, David F; Lee, Daniel W; Ren, Jiaqiang; Sabatino, Marianna; Highfill, Steven; Khuu, Hanh; Shah, Nirali N; Kaplan, Rosandra N; Fry, Terry J; Mackall, Crystal L

    2017-03-16

    Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes. Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We investigated the use of PBMC concentrates enriched for lymphocytes using elutriation for manufacturing 8 CD19- and 5 GD2-CAR T cell products. When compared to PBMC concentrates, lymphocyte-enriched elutriation fractions contained greater proportions of CD3+ and CD56+ cells and reduced proportions of CD14+ and CD15+ cells. All 13 CAR T cell products manufactured using the elutriated lymphocytes yielded sufficient quantities of transduced CAR T cells to meet clinical dose criteria. The GD2-CAR T cell products contained significantly more T cells and transduced T cells than the CD19-CAR T cell products. A comparison of the yields of CAR T cells produced from elutriated lymphocytes with the yields of CAR T cells previous produced from cells isolated from PBMC concentrates by anti-CD3/CD28 bead selection or by anti-CD3/CD28 bead selection plus plastic adherence found that greater quantities of GD2-CAR T cells were produced from elutriated lymphocytes, but not CD19-CAR T cells. Enrichment of PBMC concentrates for lymphocytes using elutriation increased the quantity of GD2-CAR T cells produced. These results provide further evidence that CAR T cell expansion is inhibited by monocytes and granulocytes.

  2. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes.

    PubMed

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F

    2015-08-18

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation.

  3. Chimeric Antigen Receptor T-cell Therapies for Multiple Myeloma.

    PubMed

    Mikkilineni, Lekha; Kochenderfer, James N

    2017-09-19

    Multiple myeloma (MM) is a nearly always incurable malignancy of plasma cells, so new approaches to treatment are needed. T-cell therapies are a promising approach for treating MM, with a mechanism of action different than those of standard MM treatments. Chimeric antigen receptors (CARs) are fusion proteins incorporating antigen-recognition domains and T-cell signaling domains. T-cells genetically engineered to express CARs can specifically recognize antigens. Success of CAR T-cells against leukemia and lymphoma has encouraged development of CAR T-cell therapies for MM. Target antigens for CARs must be expressed on malignant cells, but expression on normal cells must be absent or limited. B-cell maturation antigen (BCMA) is expressed by normal and malignant plasma cells. CAR T-cells targeting B-cell maturation antigen have demonstrated significant anti-myeloma activity in early clinical trials. Toxicities in these trials, including cytokine-release syndrome, have been similarto toxicities observed in CAR T-cell trials for leukemia. Targeting postulated CD19(+) myeloma stem cells with anti-CD19 CAR T-cells is a novel approach to MM therapy. MM antigens including CD138, CD38, signaling lymphocyte-activating molecule 7 (SLAMF7), and kappa light chain are under investigation as CAR targets. MM is genetically and phenotypically heterogeneous, so targeting of more than one antigen might often be required for effective treatment of MM with CAR T cells. Integration of CAR T cells with other myeloma therapies is an important area of future research. CAR T cell therapies for MM are at an early stage of development but have great promise to improve MM treatment. Copyright © 2017 American Society of Hematology.

  4. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes

    PubMed Central

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F.

    2015-01-01

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners—the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)—and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic—and plant and algal—lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller’s ratchet—the origin of eukaryotic recombination, or sex—might have required surprisingly little evolutionary innovation. PMID:25733873

  5. Simulations of Mineral Dust Content With CHIMERE-Dust Model

    NASA Astrophysics Data System (ADS)

    Schmechtig, C.; Marticorena, B.; Menut, L.; Bergametti, G.

    2006-12-01

    Simulations of the mineral dust cycle have been performed whith CHIMERE-Dust model over a domain that includes North Africa, the Mediterranean basin and the North Tropical Atlantic Ocean (10S-60N and 90W-90E) with a 1°x1° resolution using the ECMWF (European Center for Medium-Range Weather Forecasts) meteorological fields for two years, 2000 and 2001. As a validation, we compare the simulated dust concentration fields with photometric data from the AERONET network. From the comparisons between the simulated and measured aerosol optical depth for several stations of the Mediterranean basin, the model appears to reproduce correctly the intensity and occurrences of the dust events. Over Western Africa, the results are not as satisfying since some of the most intense dust events observed on the continent and downwind are not captured by the model. In addition, the simulated events are generally underestimated compared to the measured ones. It appears that these differences in the model performances are connected to the origin of the dust plumes. For example, dust plumes coming from Libya are well simulated while dust plumes originating from the Bodélé depression not as frequent as intense as the observations suggest. Soil properties in these two regions are comparable and typical of very erodible surfaces. We thus focused on the comparison between the ECMWF 10m wind speed fields and 10m wind speed measured at the meteorological stations located in both areas. We noticed that over Libya, the measured and ECMWF 10m wind speed are in very good agreement, while the meteorological model does not reproduce the extrema of the measured wind speed in the Bodélé depression. We found that a crude empirical correction of the 10m wind field in the Bodélé Depression significantly improve the simulations in terms of occurrence and of intensity.

  6. Development of a recombinant, chimeric tetravalent dengue vaccine candidate.

    PubMed

    Osorio, Jorge E; Partidos, Charalambos D; Wallace, Derek; Stinchcomb, Dan T

    2015-12-10

    Dengue is a significant threat to public health worldwide. Currently, there are no licensed vaccines available for dengue. Takeda Vaccines Inc. is developing a live, attenuated tetravalent dengue vaccine candidate (TDV) that consists of an attenuated DENV-2 strain (TDV-2) and three chimeric viruses containing the prM and E protein genes of DENV-1, -3 and -4 expressed in the context of the attenuated TDV-2 genome backbone (TDV-1, TDV-3, and TDV-4, respectively). TDV has been shown to be immunogenic and efficacious in nonclinical animal models. In interferon-receptor deficient mice, the vaccine induces humoral neutralizing antibody responses and cellular immune responses that are sufficient to protect from lethal challenge with DENV-1, DENV-2 or DENV-4. In non-human primates, administration of TDV induces innate immune responses as well as long lasting antibody and cellular immunity. In Phase 1 clinical trials, the safety and immunogenicity of two different formulations were assessed after intradermal or subcutaneous administration to healthy, flavivirus-naïve adults. TDV administration was generally well-tolerated independent of dose and route. The vaccine induced neutralizing antibody responses to all four DENV serotypes: after a single administration of the higher formulation, 24-67%% of the subjects seroconverted to all four DENV and >80% seroconverted to three or more viruses. In addition, TDV induced CD8(+) T cell responses to the non-structural NS1, NS3 and NS5 proteins of DENV. TDV has been also shown to be generally well tolerated and immunogenic in a Phase 2 clinical trial in dengue endemic countries in adults and children as young as 18 months. Additional clinical studies are ongoing in preparation for a Phase 3 safety and efficacy study.

  7. Buoyancy instability of homologous implosions

    DOE PAGES

    Johnson, B. M.

    2015-06-15

    With this study, I consider the hydrodynamic stability of imploding ideal gases as an idealized model for inertial confinement fusion capsules, sonoluminescent bubbles and the gravitational collapse of astrophysical gases. For oblate modes (short-wavelength incompressive modes elongated in the direction of the mean flow), a second-order ordinary differential equation is derived that can be used to assess the stability of any time-dependent flow with planar, cylindrical or spherical symmetry. Upon further restricting the analysis to homologous flows, it is shown that a monatomic gas is governed by the Schwarzschild criterion for buoyant stability. Under buoyantly unstable conditions, both entropy andmore » vorticity fluctuations experience power-law growth in time, with a growth rate that depends upon mean flow gradients and, in the absence of dissipative effects, is independent of mode number. If the flow accelerates throughout the implosion, oblate modes amplify by a factor (2C)|N0|ti, where C is the convergence ratio of the implosion, N0 is the initial buoyancy frequency and ti is the implosion time scale. If, instead, the implosion consists of a coasting phase followed by stagnation, oblate modes amplify by a factor exp(π|N0|ts), where N0 is the buoyancy frequency at stagnation and ts is the stagnation time scale. Even under stable conditions, vorticity fluctuations grow due to the conservation of angular momentum as the gas is compressed. For non-monatomic gases, this additional growth due to compression results in weak oscillatory growth under conditions that would otherwise be buoyantly stable; this over-stability is consistent with the conservation of wave action in the fluid frame. The above analytical results are verified by evolving the complete set of linear equations as an initial value problem, and it is demonstrated that oblate modes are the fastest-growing modes and that high mode numbers are required to reach this limit (Legendre mode ℓ ≳ 100

  8. Buoyancy instability of homologous implosions

    SciTech Connect

    Johnson, B. M.

    2015-06-15

    With this study, I consider the hydrodynamic stability of imploding ideal gases as an idealized model for inertial confinement fusion capsules, sonoluminescent bubbles and the gravitational collapse of astrophysical gases. For oblate modes (short-wavelength incompressive modes elongated in the direction of the mean flow), a second-order ordinary differential equation is derived that can be used to assess the stability of any time-dependent flow with planar, cylindrical or spherical symmetry. Upon further restricting the analysis to homologous flows, it is shown that a monatomic gas is governed by the Schwarzschild criterion for buoyant stability. Under buoyantly unstable conditions, both entropy and vorticity fluctuations experience power-law growth in time, with a growth rate that depends upon mean flow gradients and, in the absence of dissipative effects, is independent of mode number. If the flow accelerates throughout the implosion, oblate modes amplify by a factor (2C)|N0|ti, where C is the convergence ratio of the implosion, N0 is the initial buoyancy frequency and ti is the implosion time scale. If, instead, the implosion consists of a coasting phase followed by stagnation, oblate modes amplify by a factor exp(π|N0|ts), where N0 is the buoyancy frequency at stagnation and ts is the stagnation time scale. Even under stable conditions, vorticity fluctuations grow due to the conservation of angular momentum as the gas is compressed. For non-monatomic gases, this additional growth due to compression results in weak oscillatory growth under conditions that would otherwise be buoyantly stable; this over-stability is consistent with the conservation of wave action in the fluid frame. The above analytical results are verified by evolving the complete set of linear equations as an initial value problem, and it is demonstrated that oblate modes are the fastest

  9. Chimeric mouse-human IgG1 antibody that can mediate lysis of cancer cells.

    PubMed Central

    Liu, A Y; Robinson, R R; Hellström, K E; Murray, E D; Chang, C P; Hellström, I

    1987-01-01

    A chimeric mouse-human antibody has been created that recognizes an antigen found on the surface of cells from many carcinomas. Immunoglobulin constant (C) domains of the mouse monoclonal antibody L6, C gamma 2a and C kappa, were substituted by the human C gamma 1 and C kappa by recombining cDNA modules encoding variable or C domains. The cDNA constructs were transfected into lymphoid cells for antibody production. The chimeric antibody and mouse L6 antibody bound to carcinoma cells with equal affinity and mediated complement-dependent cytolysis. In the presence of human effector cells, the chimeric antibody gave antibody-dependent cellular cytotoxicity at 100 times lower concentration than that needed for the mouse L6 antibody. The chimeric antibody, but not the mouse L6 antibody, is effective against a melanoma line expressing small amounts of the L6 antigen. The findings point to the usefulness of the chimeric antibody approach for obtaining agents with strong antitumor activity for possible therapeutic use in man. PMID:3106970

  10. Production of a neutralizing mouse-human chimeric antibody against botulinum neurotoxin serotype E.

    PubMed

    Mukamoto, Masafumi; Maeda, Hiroaki; Kohda, Tomoko; Nozaki, Chikateru; Takahashi, Motohide; Kozaki, Shunji

    2013-01-01

    A mouse-human chimeric antibody that can neutralize botulinum neurotoxin serotype E (BoNT/E) was developed. Variable regions of heavy and light chains obtained using a mouse hybridoma clone (E9-4) cDNA, which was selected on the basis of neutralizing activity against BoNT/E, were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and a mouse-human chimeric antibody (EC94) was purified to examine binding and neutralizing activity against BoNT/E. EC94 exhibited the same levels of binding activities against BoNT/E as those of a parent mouse monoclonal antibody and neutralized more than 4,000 LD(50)/mg antibody. This chimeric antibody seems to be a useful candidate for infant botulism in which the use of passive immunotherapy is not planned so as to avoid serious events such as anaphylactic shock. We designed shuffling chimeric antibodies with replacement of V(H) or V(L) of EC94 with that of a chimeric antibody (AC24) that possessed neutralizing activity against BoNT/A. These shuffling antibodies did not exhibit neutralizing activity against either BoNT/E or BoNT/A.

  11. Interleukin 2-Bax: a novel prototype of human chimeric proteins for targeted therapy.

    PubMed

    Aqeilan, R; Yarkoni, S; Lorberboum-Galski, H

    1999-08-27

    During the past few years many chimeric proteins have been developed to target and kill cells expressing specific surface molecules. Generally, these molecules carry a bacterial or plant toxin that destroys the unwanted cells. The major obstacle in the clinical application of such chimeras is their immunogenicity and non-specific toxicity. We have developed a new generation of chimeric proteins, taking advantage of apoptosis-inducing proteins, such as the human Bax protein, as novel killing components. The first prototype chimeric protein, IL2-Bax, directed toward IL2R-expressing cells, was constructed, expressed in Escherichia coli and partially purified. IL2-Bax increased the population of apoptotic cells in a variety of target T cell lines, as well as in human fresh PHA-activated lymphocytes, in a dose-dependent manner and had no effect on cells lacking IL2R expression. The IL2-Bax chimera represents an innovative approach for constructing chimeric proteins comprising a molecule that binds a specific cell type and an apoptosis-inducing protein. Such new chimeric proteins could be used for targeted treatment of human diseases.

  12. Induction of Pluripotent Protective Immunity Following Immunisation with a Chimeric Vaccine against Human Cytomegalovirus

    PubMed Central

    Zhong, Jie; Rist, Michael; Cooper, Leanne; Smith, Corey; Khanna, Rajiv

    2008-01-01

    Based on the life-time cost to the health care system, the Institute of Medicine has assigned the highest priority for a vaccine to control human cytomegalovirus (HCMV) disease in transplant patients and new born babies. In spite of numerous attempts successful licensure of a HCMV vaccine formulation remains elusive. Here we have developed a novel chimeric vaccine strategy based on a replication-deficient adenovirus which encodes the extracellular domain of gB protein and multiple HLA class I & II-restricted CTL epitopes from HCMV as a contiguous polypeptide. Immunisation with this chimeric vaccine consistently generated strong HCMV-specific CD8+ and CD4+ T-cells which co-expressed IFN-γ and TNF-α, while the humoral response induced by this vaccine showed strong virus neutralizing capacity. More importantly, immunization with adenoviral chimeric vaccine also afforded protection against challenge with recombinant vaccinia virus encoding HCMV antigens and this protection was associated with the induction of a pluripotent antigen-specific cellular and antibody response. Furthermore, in vitro stimulation with this adenoviral chimeric vaccine rapidly expanded multiple antigen-specific human CD8+ and CD4+ T-cells from healthy virus carriers. These studies demonstrate that the adenovirus chimeric HCMV vaccine provides an excellent platform for reconstituting protective immunity to prevent HCMV diseases in different clinical settings. PMID:18806877

  13. Reversible Heat-Induced Inactivation of Chimeric β-Glucuronidase in Transgenic Plants1

    PubMed Central

    Almoguera, Concepción; Rojas, Anabel; Jordano, Juan

    2002-01-01

    We compared the expression patterns in transgenic tobacco (Nicotiana tabacum) of two chimeric genes: a translational fusion to β-glucuronidase (GUS) and a transcriptional fusion, both with the same promoter and 5′-flanking sequences of Ha hsp17.7 G4, a small heat shock protein (sHSP) gene from sunflower (Helianthus annuus). We found that immediately after heat shock, the induced expression from the two fusions in seedlings was similar, considering chimeric mRNA or GUS protein accumulation. Surprisingly, we discovered that the chimeric GUS protein encoded by the translational fusion was mostly inactive in such conditions. We also found that this inactivation was fully reversible. Thus, after returning to control temperature, the GUS activity was fully recovered without substantial changes in GUS protein accumulation. In contrast, we did not find differences in the in vitro heat inactivation of the respective GUS proteins. Insolubilization of the chimeric GUS protein correlated with its inactivation, as indicated by immunoprecipitation analyses. The inclusion in another chimeric gene of the 21 amino-terminal amino acids from a different sHSP lead to a comparable reversible inactivation. That effect not only illustrates unexpected post-translational problems, but may also point to sequences involved in interactions specific to sHSPs and in vivo heat stress conditions. PMID:12011363

  14. Reversible heat-induced inactivation of chimeric beta-glucuronidase in transgenic plants.

    PubMed

    Almoguera, Concepción; Rojas, Anabel; Jordano, Juan

    2002-05-01

    We compared the expression patterns in transgenic tobacco (Nicotiana tabacum) of two chimeric genes: a translational fusion to beta-glucuronidase (GUS) and a transcriptional fusion, both with the same promoter and 5'-flanking sequences of Ha hsp17.7 G4, a small heat shock protein (sHSP) gene from sunflower (Helianthus annuus). We found that immediately after heat shock, the induced expression from the two fusions in seedlings was similar, considering chimeric mRNA or GUS protein accumulation. Surprisingly, we discovered that the chimeric GUS protein encoded by the translational fusion was mostly inactive in such conditions. We also found that this inactivation was fully reversible. Thus, after returning to control temperature, the GUS activity was fully recovered without substantial changes in GUS protein accumulation. In contrast, we did not find differences in the in vitro heat inactivation of the respective GUS proteins. Insolubilization of the chimeric GUS protein correlated with its inactivation, as indicated by immunoprecipitation analyses. The inclusion in another chimeric gene of the 21 amino-terminal amino acids from a different sHSP lead to a comparable reversible inactivation. That effect not only illustrates unexpected post-translational problems, but may also point to sequences involved in interactions specific to sHSPs and in vivo heat stress conditions.

  15. The homologous recombination system of Ustilago maydis.

    PubMed

    Holloman, William K; Schirawski, Jan; Holliday, Robin

    2008-08-01

    Homologous recombination is a high fidelity, template-dependent process that is used in repair of damaged DNA, recovery of broken replication forks, and disjunction of homologous chromosomes in meiosis. Much of what is known about recombination genes and mechanisms comes from studies on baker's yeast. Ustilago maydis, a basidiomycete fungus, is distant evolutionarily from baker's yeast and so offers the possibility of gaining insight into recombination from an alternative perspective. Here we have surveyed the genome of U. maydis to determine the composition of its homologous recombination system. Compared to baker's yeast, there are fundamental differences in the function as well as in the repertoire of dedicated components. These include the use of a BRCA2 homolog and its modifier Dss1 rather than Rad52 as a mediator of Rad51, the presence of only a single Rad51 paralog, and the absence of Dmc1 and auxiliary meiotic proteins.

  16. The homologous recombination system of Ustilago maydis

    PubMed Central

    Holloman, William K.; Schirawski, Jan; Holliday, Robin

    2008-01-01

    Homologous recombination is a high fidelity, template-dependent process that is used in repair of damaged DNA, recovery of broken replication forks, and disjunction of homologous chromosomes in meiosis. Much of what is known about recombination genes and mechanisms comes from studies on baker's yeast. Ustilago maydis, a basidiomycete fungus, is distant evolutionarily from baker's yeast and so offers the possibility of gaining insight into recombination from an alternative perspective. Here we have surveyed the genome of Ustilago maydis to determine the composition of its homologous recombination system. Compared to baker's yeast, there are fundamental differences in the function as well as in the repertoire of dedicated components. These include the use of a BRCA2 homolog and its modifier Dss1 rather than Rad52 as a mediator of Rad51, the presence of only a single Rad51 paralog, and the absence of Dmc1 and auxiliary meiotic proteins. PMID:18502156

  17. Homology class of a Lagrangian Klein bottle

    NASA Astrophysics Data System (ADS)

    Nemirovski, Stefan Yu

    2009-08-01

    It is shown that an embedded Lagrangian Klein bottle realises a non-zero mod 2 homology class in a compact symplectic four-manifold (X,\\omega) such that c_1(X,\\omega)\\cdot \\lbrack \\omega \\rbrack > 0.

  18. Persistent homology analysis of phase transitions

    NASA Astrophysics Data System (ADS)

    Donato, Irene; Gori, Matteo; Pettini, Marco; Petri, Giovanni; De Nigris, Sarah; Franzosi, Roberto; Vaccarino, Francesco

    2016-05-01

    Persistent homology analysis, a recently developed computational method in algebraic topology, is applied to the study of the phase transitions undergone by the so-called mean-field XY model and by the ϕ4 lattice model, respectively. For both models the relationship between phase transitions and the topological properties of certain submanifolds of configuration space are exactly known. It turns out that these a priori known facts are clearly retrieved by persistent homology analysis of dynamically sampled submanifolds of configuration space.

  19. Dualities in Persistent (Co)Homology

    SciTech Connect

    de Silva, Vin; Morozov, Dmitriy; Vejdemo-Johansson, Mikael

    2011-09-16

    We consider sequences of absolute and relative homology and cohomology groups that arise naturally for a filtered cell complex. We establishalgebraic relationships between their persistence modules, and show that they contain equivalent information. We explain how one can use the existingalgorithm for persistent homology to process any of the four modules, and relate it to a recently introduced persistent cohomology algorithm. Wepresent experimental evidence for the practical efficiency of the latter algorithm.

  20. Persistent homology analysis of phase transitions.

    PubMed

    Donato, Irene; Gori, Matteo; Pettini, Marco; Petri, Giovanni; De Nigris, Sarah; Franzosi, Roberto; Vaccarino, Francesco

    2016-05-01

    Persistent homology analysis, a recently developed computational method in algebraic topology, is applied to the study of the phase transitions undergone by the so-called mean-field XY model and by the ϕ^{4} lattice model, respectively. For both models the relationship between phase transitions and the topological properties of certain submanifolds of configuration space are exactly known. It turns out that these a priori known facts are clearly retrieved by persistent homology analysis of dynamically sampled submanifolds of configuration space.

  1. Preserved irradiated homologous cartilage for orbital reconstruction

    SciTech Connect

    Linberg, J.V.; Anderson, R.L.; Edwards, J.J.; Panje, W.R.; Bardach, J.

    1980-07-01

    Human costal cartilage is an excellent implant material for orbital and periorbital reconstruction because of its light weight, strength, homogeneous consistency and the ease with which it can be carved. Its use has been limited by the necessity of a separate surgical procedure to obtain the material. Preserved irradiated homologous cartilage has been shown to have almost all the autogenous cartilage and is convenient to use. Preserved irradiated homologous cartilage transplants do not elicit rejection reactions, resist infection and rarely undergo absorption.

  2. On the hodological criterion for homology.

    PubMed

    Faunes, Macarena; Francisco Botelho, João; Ahumada Galleguillos, Patricio; Mpodozis, Jorge

    2015-01-01

    Owen's pre-evolutionary definition of a homolog as "the same organ in different animals under every variety of form and function" and its redefinition after Darwin as "the same trait in different lineages due to common ancestry" entail the same heuristic problem: how to establish "sameness."Although different criteria for homology often conflict, there is currently a generalized acceptance of gene expression as the best criterion. This gene-centered view of homology results from a reductionist and preformationist concept of living beings. Here, we adopt an alternative organismic-epigenetic viewpoint, and conceive living beings as systems whose identity is given by the dynamic interactions between their components at their multiple levels of composition. We posit that there cannot be an absolute homology criterion, and instead, homology should be inferred from comparisons at the levels and developmental stages where the delimitation of the compared trait lies. In this line, we argue that neural connectivity, i.e., the hodological criterion, should prevail in the determination of homologies between brain supra-cellular structures, such as the vertebrate pallium.

  3. Homolog pairing and segregation in Drosophila meiosis.

    PubMed

    McKee, B D

    2009-01-01

    Pairing of homologous chromosomes is fundamental to their reliable segregation during meiosis I and thus underlies sexual reproduction. In most eukaryotes homolog pairing is confined to prophase of meiosis I and is accompanied by frequent exchanges, known as crossovers, between homologous chromatids. Crossovers give rise to chiasmata, stable interhomolog connectors that are required for bipolar orientation (orientation to opposite poles) of homologs during meiosis I. Drosophila is unique among model eukaryotes in exhibiting regular homolog pairing in mitotic as well as meiotic cells. I review the results of recent molecular studies of pairing in both mitosis and meiosis in Drosophila. These studies show that homolog pairing is continuous between pre-meiotic mitosis and meiosis but that pairing frequencies and patterns are altered during the mitotic-meiotic transition. They also show that, with the exception of X-Y pairing in male meiosis, which is mediated specifically by the 240-bp rDNA spacer repeats, chromosome pairing is not restricted to specific sites in either mitosis or meiosis. Instead, virtually all chromosome regions, both heterochromatic and euchromatic, exhibit autonomous pairing capacity. Mutations that reduce the frequencies of both mitotic and meiotic pairing have been recently described, but no mutations that abolish pairing completely have been discovered, and the genetic control of pairing in Drosophila remains to be elucidated.

  4. On the hodological criterion for homology

    PubMed Central

    Faunes, Macarena; Francisco Botelho, João; Ahumada Galleguillos, Patricio; Mpodozis, Jorge

    2015-01-01

    Owen's pre-evolutionary definition of a homolog as “the same organ in different animals under every variety of form and function” and its redefinition after Darwin as “the same trait in different lineages due to common ancestry” entail the same heuristic problem: how to establish “sameness.”Although different criteria for homology often conflict, there is currently a generalized acceptance of gene expression as the best criterion. This gene-centered view of homology results from a reductionist and preformationist concept of living beings. Here, we adopt an alternative organismic-epigenetic viewpoint, and conceive living beings as systems whose identity is given by the dynamic interactions between their components at their multiple levels of composition. We posit that there cannot be an absolute homology criterion, and instead, homology should be inferred from comparisons at the levels and developmental stages where the delimitation of the compared trait lies. In this line, we argue that neural connectivity, i.e., the hodological criterion, should prevail in the determination of homologies between brain supra-cellular structures, such as the vertebrate pallium. PMID:26157357

  5. Equivalent perceptual asymmetries for free viewing of positive and negative emotional expressions in chimeric faces.

    PubMed

    Christman, S D; Hackworth, M D

    1993-06-01

    Research employing chimeric stimuli (in which smiling and neutral half-faces are paired) has demonstrated greater influence of the left half-face in determining perceived intensity of expression. To date, no studies have examined how emotional expressions other than happiness are perceived in this format. Right-handed subjects viewed chimeric faces depicting both positive (happiness, pleasant surprise) and negative (sadness, anger) emotions in a free vision task. Results indicated a left half-face bias for all four emotions, supporting the hypothesis of a greater right hemisphere role in emotional perception. The lack of differences in strength of left half-face bias as a function of the specific emotion depicted suggests that results obtained with typical chimeric half-face paradigms can be generalized to emotions other than happiness.

  6. CHIMERIC SINDBIS/EASTERN EQUINE ENCEPHALITIS VACCINE CANDIDATES ARE HIGHLY ATTENUATED AND IMMUNOGENIC IN MICE

    PubMed Central

    Wang, Eryu; Petrakova, Olga; Adams, A. Paige; Aguilar, Patricia V.; Kang, Wenli; Paessler, Slobodan; Volk, Sara M.; Frolov, Ilya; Weaver, Scott C.

    2007-01-01

    We developed chimeric Sindbis (SINV)/Eastern equine encephalitis (EEEV) viruses and investigated their potential for use as live virus vaccines against EEEV. One vaccine candidate contained structural protein genes from a typical North American EEEV strain, while the other had structural proteins from a naturally attenuated Brazilian isolate. Both chimeric viruses replicated efficiently in mammalian and mosquito cell cultures and were highly attenuated in mice. Vaccinated mice did not develop detectable disease or viremia, but developed high titers of neutralizing antibodies. Upon challenge with EEEV, mice vaccinated with >104PFU of the chimeric viruses were completely protected from disease. These findings support the potential use of these SIN/EEEV chimeras as safe and effective vaccines. PMID:17904699

  7. Functionalization of scaffolds with chimeric anti-BMP-2 monoclonal antibodies for osseous regeneration

    PubMed Central

    Ansari, Sahar; Moshaverinia, Alireza; Pi, Sung Hee; Han, Alexander; Abdelhamid, Alaa I.; Zadeh, Homayoun H.

    2013-01-01

    Recent studies have demonstrated the ability of murine anti-BMP-2 monoclonal antibodies (mAb) immobilized on an absorbable collagen sponge (ACS) to mediate de novo bone formation, a process termed antibody mediated osseous regeneration (AMOR). The objectives of this study were to assess the efficacy of a newly generated chimeric anti-BMP-2 mAb in mediating AMOR, as well as to evaluate the suitability of different biomaterials as scaffolds to participate in AMOR. Chimeric anti-BMP-2 mAb was immobilized on 4 biomaterials, namely, titanium microbeads (Ti), alginate hydrogel, macroporous biphasic calcium phosphate (MBCP) and ACS, followed by surgical implantation into rat critical-size calvarial defects. Animals were sacrificed after 8 weeks and the degree of bone fill was assessed using micro-CT and histomorphometry. Results demonstrated local persistence of chimeric anti-BMP-2 mAb up to 8 weeks, as well as significant de novo bone regeneration in sites implanted with chimeric anti-BMP-2 antibody immobilized on each of the 4 scaffolds. Ti and MBCP showed the highest volume of bone regeneration, presumably due to their resistance to compression. Alginate and ACS also mediated de novo bone formation, though significant volumetric shrinkage was noted. In vitro assays demonstrated cross-reactivity of chimeric anti-BMP-2 mAb with BMP-4 and BMP-7. Immune complex of anti-BMP-2 mAb with BMP-2 induced osteogenic differentiation of C2C12 cells in vitro, involving expression of RUNX2 and phosphorylation of Smad1. The present data demonstrated the ability of chimeric anti- BMP-2 mAb to functionalize different biomaterial with varying characteristics to mediate osteogenesis. PMID:24055525

  8. Structure-function analysis of peroxisomal ATP-binding cassette transporters using chimeric dimers.

    PubMed

    Geillon, Flore; Gondcaille, Catherine; Charbonnier, Soëli; Van Roermund, Carlo W; Lopez, Tatiana E; Dias, Alexandre M M; Pais de Barros, Jean-Paul; Arnould, Christine; Wanders, Ronald J; Trompier, Doriane; Savary, Stéphane

    2014-08-29

    ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Structure-Function Analysis of Peroxisomal ATP-binding Cassette Transporters Using Chimeric Dimers*

    PubMed Central

    Geillon, Flore; Gondcaille, Catherine; Charbonnier, Soëli; Van Roermund, Carlo W.; Lopez, Tatiana E.; Dias, Alexandre M. M.; Pais de Barros, Jean-Paul; Arnould, Christine; Wanders, Ronald J.; Trompier, Doriane; Savary, Stéphane

    2014-01-01

    ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters. PMID:25043761

  10. Chimeric Antigen Receptor–Modified T Cells for Acute Lymphoid Leukemia

    PubMed Central

    Barrett, David; Aplenc, Richard; Porter, David L.; Rheingold, Susan R.; Teachey, David T.; Chew, Anne; Hauck, Bernd; Wright, J. Fraser; Milone, Michael C.; Levine, Bruce L.; June, Carl H.

    2014-01-01

    Summary Chimeric antigen receptor–modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre–B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×106 to 1.2×107 CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce anti-leukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor–modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL. PMID:23527958

  11. Generation and developmental characteristics of porcine tetraploid embryos and tetraploid/diploid chimeric embryos.

    PubMed

    He, Wenteng; Kong, Qingran; Shi, Yongqian; Xie, Bingteng; Jiao, Mingxia; Huang, Tianqing; Guo, Shimeng; Hu, Kui; Liu, Zhonghua

    2013-10-01

    The aim of this study was to optimize electrofusion conditions for generating porcine tetraploid (4n) embryos and produce tetraploid/diploid (4n/2n) chimeric embryos. Different electric field intensities were tested and 2 direct current (DC) pulses of 0.9 kV/cm for 30 μs was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos. The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos, reached 85.4% and 28.5%, respectively. 68.18% of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization (FISH). Although the number of blastomeres in 4n blastocysts was significantly lower than in 2n blastocysts (P<0.05), there was no significant difference in developmental rates of blastocysts between 2n and 4n embryos (P>0.05), suggesting that the blastocyst forming capacity in 4n embryos is similar to those in 2n embryos. Moreover, 4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos. We found that the developmental rate and cell number of blastocysts of 4-cell (4n)/4-cell (2n) chimeric embryos were significantly higher than those of 2-cell (4n)/4-cell (2n), 4-cell (4n)/8-cell (2n), 4-cell (4n)/2-cell (2n) chimeric embryos (P<0.05). Consistent with mouse chimeras, the majority of 4n cells contribute to the trophectoderm (TE), while the 2n cells are mainly present in the inner cell mass (ICM) of porcine 4n/2n chimeric embryos. Our study established a feasible and efficient approach to produce porcine 4n embryos and 4n/2n chimeric embryos.

  12. Bone marrow cell migration to the heart in a chimeric mouse model of acute chagasic disease

    PubMed Central

    Irion, Camila Iansen; Paredes, Bruno Diaz; Brasil, Guilherme Visconde; da Cunha, Sandro Torrentes; Paula, Luis Felipe; Carvalho, Alysson Roncally; de Carvalho, Antonio Carlos Campos; Carvalho, Adriana Bastos; Goldenberg, Regina Coeli dos Santos

    2017-01-01

    BACKGROUND Chagas disease is a public health problem caused by infection with the protozoan Trypanosoma cruzi. There is currently no effective therapy for Chagas disease. Although there is some evidence for the beneficial effect of bone marrow-derived cells in chagasic disease, the mechanisms underlying their effects in the heart are unknown. Reports have suggested that bone marrow cells are recruited to the chagasic heart; however, studies using chimeric mouse models of chagasic cardiomyopathy are rare. OBJECTIVES The aim of this study was to investigate the migration of bone marrow cells to the heart after T. cruzi infection in a model of chagasic disease in chimeric mice. METHODS To obtain chimerical mice, wild-type (WT) C57BL6 mice were exposed to full body irradiation (7 Gy), causing bone marrow ablation. Then, bone marrow cells from green fluorescent protein (GFP)-transgenic mice were infused into the mice. Graft effectiveness was confirmed by flow cytometry. Experimental mice were divided into four groups: (i) infected chimeric (iChim) mice; (ii) infected WT (iWT) mice, both of which received 3 × 104 trypomastigotes of the Brazil strain; (iii) non-infected chimeric (Chim) mice; and (iv) non-infected WT mice. FINDINGS At one-month post-infection, iChim and iWT mice showed first degree atrioventricular block with decreased heart rate and treadmill exercise parameters compared to those in the non-infected groups. MAIN CONCLUSIONS iChim mice showed an increase in parasitaemia, myocarditis, and the presence of amastigote nests in the heart tissue compared to iWT mice. Flow cytometry analysis did not detect haematopoietic progenitor cells in the hearts of infected mice. Furthermore, GFP+ cardiomyocytes were not detected in the tissues of chimeric mice. PMID:28767980

  13. 78 FR 13691 - Prospective Grant of Exclusive License: The Development of m971 and m972 Chimeric Antigen...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-28

    ... m971 and m972 Chimeric Antigen Receptors (CARs) for the Treatment of B Cell Malignancies AGENCY... worldwide, and the field of use may be limited to: Treatment of B cell malignancies that express CD22 on their cell surface using chimeric antigen receptors which contain the m971 or m972 antibody...

  14. Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties

    USDA-ARS?s Scientific Manuscript database

    The development of a spider silk manufacturing process is of great interest. piggyBac vectors were used to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk prote...

  15. Engineering and characterization of a chimeric anti-platelet glycoprotein Ibalpha monoclonal antibody and preparation of its Fab fragment.

    PubMed

    Yang, Jianfeng; Ji, Shundong; Dong, Ningzheng; Zhao, Yiming; Ruan, Changgeng

    2010-04-01

    Glycoprotein Ibalpha (GPIbalpha) is a platelet-specific membrane protein. It mediates platelet adhesion to collagen exposed at the vascular injury site by binding to von Willebrand factor (VWF) in plasma. This process is crucial for arterial thrombus formation. Blocking interaction between GPIbalpha and VWF may prevent platelet adhesion and thrombus formation. We previously generated a high affinity monoclonal antibody against human platelet GPIbalpha, SZ2, which inhibits both ristocetin- and botrocetin-induced platelet aggregation in vitro. To convert SZ2 into mouse/human chimeric antibody for anti-platelet therapy in humans, in this study, we constructed a mouse/human chimeric antibody derived from the hybridoma cells producing murine antibody against platelet glycoprotein Ibalpha, conducted its expression in dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cells, and prepared its chimeric Fab fragment. Results from ELISA and Western blot analysis showed that the chimeric antibody was secreted from the cells and that the heavy and light chains were assembled correctly. Flow cytometry analysis confirmed specific binding of the chimeric antibody to the GPIb-expressing CHO cells. In vitro functional studies revealed that the chimeric antibody and its Fab fragment prevented platelet adhesion to VWF under high shear stress and inhibited ristocetin-induced platelet aggregation in a dose-dependent manner. These results demonstrated that the chimeric antibody was successfully engineered and suggested that the Fab fragment of chimeric antibody against GPIbalpha is a promising therapeutic antibody more suitable for prevention and treatment of human arterial thrombosis.

  16. The impact of chimerism in DNA-based forensic sex determination analysis.

    PubMed

    George, Renjith; Donald, Preethy Mary; Nagraj, Sumanth Kumbargere; Idiculla, Jose Joy; Hj Ismail, Rashid

    2013-01-01

    Sex determination is the most important step in personal identification in forensic investigations. DNA-based sex determination analysis is comparatively more reliable than the other conventional methods of sex determination analysis. Advanced technology like real-time polymerase chain reaction (PCR) offers accurate and reproducible results and is at the level of legal acceptance. But still there are situations like chimerism where an individual possess both male and female specific factors together in their body. Sex determination analysis in such cases can give erroneous results. This paper discusses the phenomenon of chimerism and its impact on sex determination analysis in forensic investigations.

  17. Thermal stability of chimeric isopropylmalate dehydrogenase genes constructed from a thermophile and a mesophile.

    PubMed

    Numata, K; Muro, M; Akutsu, N; Nosoh, Y; Yamagishi, A; Oshima, T

    1995-01-01

    Chimeric isopropylmalate dehydrogenases were constructed by connecting the genes isolated from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis. These genes were expressed in Escherichia coli. The enzymes were purified and analysed. Enzymes of T.thermophilus and B.subtilis and chimeric enzymes showed similar enzymological characteristics except for thermal stability. The stability of each enzyme was approximately proportional to the content of the amino acid sequence from the T.thermophilus enzyme. The results suggested that amino acid residues contributing the thermal stability distribute themselves, in general, evenly at least in the N-terminal half of the amino acid sequence of T.thermophilus isopropylmalate dehydrogenase.

  18. TLR agonists abrogate costimulation blockade-induced mixed chimerism and transplantation tolerance

    PubMed Central

    Miller, David M.; Thornley, Thomas; Pearson, Todd; Yamazaki, Masahiro; Brehm, Michael A.; Rossini, Aldo A.; Greiner, Dale L.

    2008-01-01

    We investigated the mechanisms by which toll-like receptor (TLR) agonists affect the induction of mixed chimerism and skin allograft survival in mice treated with costimulation blockade (CB). We report that TLR agonists prevent the generation of mixed chimerism by breaking tolerance in the alloreactive CD4+ and CD8+ T cell compartments, and that type I interferon (IFN) is important in this process. Understanding how environmental perturbations affect CB-induced transplantation tolerance may lead to more effective regimens that can be used as an approach for the treatment of type 1 diabetes, for which the transplantation of pancreatic islets is a promising therapy. PMID:19120285

  19. Interspecies chimeric complementation for the generation of functional human tissues and organs in large animal hosts.

    PubMed

    Wu, Jun; Izpisua Belmonte, Juan Carlos

    2016-06-01

    The past decade's rapid progress in human pluripotent stem cell (hPSC) research has generated hope for meeting the rising demand of organ donation, which remains the only effective cure for end-stage organ failure, a major cause of death worldwide. Despite the potential, generation of transplantable organs from hPSCs using in vitro differentiation is far-fetched. An in vivo interspecies chimeric complementation strategy relying on chimeric-competent hPSCs and zygote genome editing provides an auspicious alternative for providing unlimited organ source for transplantation.

  20. The impact of chimerism in DNA-based forensic sex determination analysis

    PubMed Central

    George, Renjith; Donald, Preethy Mary; Nagraj, Sumanth Kumbargere; Idiculla, Jose Joy; Hj Ismail, Rashid

    2013-01-01

    Sex determination is the most important step in personal identification in forensic investigations. DNA-based sex determination analysis is comparatively more reliable than the other conventional methods of sex determination analysis. Advanced technology like real-time polymerase chain reaction (PCR) offers accurate and reproducible results and is at the level of legal acceptance. But still there are situations like chimerism where an individual possess both male and female specific factors together in their body. Sex determination analysis in such cases can give erroneous results. This paper discusses the phenomenon of chimerism and its impact on sex determination analysis in forensic investigations. PMID:23785258

  1. Investigating Homology between Proteins using Energetic Profiles

    PubMed Central

    Wrabl, James O.; Hilser, Vincent J.

    2010-01-01

    Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may

  2. Investigating homology between proteins using energetic profiles.

    PubMed

    Wrabl, James O; Hilser, Vincent J

    2010-03-26

    Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may

  3. Homologs of Breast Cancer Genes in Plants

    PubMed Central

    Trapp, Oliver; Seeliger, Katharina; Puchta, Holger

    2011-01-01

    Since the initial discovery of genes involved in hereditary breast cancer in humans, a vast wealth of information has been published. Breast cancer proteins were shown to work as tumor suppressors primarily through their involvement in DNA-damage repair. Surprisingly, homologs of these genes can be found in plant genomes, as well. Here, we want to give an overview of the identification and characterization of the biological roles of these proteins, in plants. In addition to the conservation of their function in DNA repair, new plant-specific characteristics have been revealed. BRCA1 is required for the efficient repair of double strand breaks (DSB) by homologous recombination in somatic cells of the model plant Arabidopsis thaliana. Bioinformatic analysis indicates that, whereas most homologs of key components of the different mammalian BRCA1 complexes are present in plant genomes, homologs of most factors involved in the recruitment of BRCA1 to the DSB cannot be identified. Thus, it is not clear at the moment whether differences exist between plants and animals at this important step. The most conserved region of BRCA1 and BARD1 homologs in plants is a PHD domain which is absent in mammals and which, in AtBARD1, might be involved in the transcriptional regulation of plant development. The presence of a plant-specific domain prompted us to reevaluate the current model for the evolution of BRCA1 homologs and to suggest a new hypothesis, in which we postulate that plant BRCA1 and BARD1 have one common predecessor that gained a PHD domain before duplication. Furthermore, work in Arabidopsis demonstrates that – as in animals – BRCA2 homologs are important for meiotic DNA recombination. Surprisingly, recent research has revealed that AtBRCA2 also has an important role in systemic acquired resistance. In Arabidopsis, BRCA2 is involved in the transcriptional regulation of pathogenesis-related (PR) genes via its interaction with the strand exchange protein RAD51. PMID

  4. Tumor-Triggered Geometrical Shape Switch of Chimeric Peptide for Enhanced in Vivo Tumor Internalization and Photodynamic Therapy.

    PubMed

    Han, Kai; Zhang, Jin; Zhang, Weiyun; Wang, Shibo; Xu, Luming; Zhang, Chi; Zhang, Xianzheng; Han, Heyou

    2017-03-28

    Geometrical shape of nanoparticles plays an important role in cellular internalization. However, the applicability in tumor selective therapeutics is still scarcely reported. In this article, we designed a tumor extracellular acidity-responsive chimeric peptide with geometrical shape switch for enhanced tumor internalization and photodynamic therapy. This chimeric peptide could self-assemble into spherical nanoparticles at physiological condition. While at tumor extracellular acidic microenvironment, chimeric peptide underwent detachment of acidity-sensitive 2,3-dimethylmaleic anhydride groups. The subsequent recovery of ionic complementarity between chimeric peptides resulted in formation of rod-like nanoparticles. Both in vitro and in vivo studies demonstrated that this acidity-triggered geometrical shape switch endowed chimeric peptide with accelerated internalization in tumor cells, prolonged accumulation in tumor tissue, enhanced photodynamic therapy, and minimal side effects. Our results suggested that fusing tumor microenvironment with geometrical shape switch should be a promising strategy for targeted drug delivery.

  5. Follow up of hemopoietic chimerism in individuals given allogeneic hemopoietic stem cell allografts using an immunosuppressive, non-myeloablative conditioning regimen: a prospective study in a single institution.

    PubMed

    Ruiz-Argüelles, Guillermo J; López-Martíneza, Briceida; Santellán-Olea, Ma Rayo; Abreu-Díaz, Glexsy; Reyes-Núñez, Virginia; Ruiz-Argüelles, Alejandro; Garcés-Eisele, Javier

    2002-07-01

    Thirty consecutive patients were given non-myeloablative stem cell transplants (NST) and posttransplant chimerism was studied by several methods. In 16 individuals definitive proofs of chimerism have been shown: In 10 cases sex chimerism, in 7 cases chimerism shown by means of microsatellites, in 4 cases ABO chimerism, in two cases Rh chimerism and in one HLA-DR chimerism. In addition, in 9 individuals the disappearance of the molecular marker of the leukemia is an indirect evidence of the chimerism, as well as the presence of graft versus host disease (GVHD) in 17 allografted patients. Only in 6 patients no evidence of chimerism could be shown; all of them died as a result of either persistent or relapsing malignancy. Since the early patterns of chimerism may be predictive of either GVHD or graft loss in NST and, since therapeutic intervention (such as donor lymphocytes infusions) is based in the patterns of chimerism, it is possible that chimerism studies in these types of allografts should be ideally done more frequently than in conventional allotransplants.

  6. Chimeric Plant Calcium/Calmodulin-Dependent Protein Kinase Gene with a Neural Visinin-Like Calcium-Binding Domain

    NASA Technical Reports Server (NTRS)

    Patil, Shameekumar; Takezawa, D.; Poovaiah, B. W.

    1995-01-01

    Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

  7. Homological scaffolds of brain functional networks

    PubMed Central

    Petri, G.; Expert, P.; Turkheimer, F.; Carhart-Harris, R.; Nutt, D.; Hellyer, P. J.; Vaccarino, F.

    2014-01-01

    Networks, as efficient representations of complex systems, have appealed to scientists for a long time and now permeate many areas of science, including neuroimaging (Bullmore and Sporns 2009 Nat. Rev. Neurosci. 10, 186–198. (doi:10.1038/nrn2618)). Traditionally, the structure of complex networks has been studied through their statistical properties and metrics concerned with node and link properties, e.g. degree-distribution, node centrality and modularity. Here, we study the characteristics of functional brain networks at the mesoscopic level from a novel perspective that highlights the role of inhomogeneities in the fabric of functional connections. This can be done by focusing on the features of a set of topological objects—homological cycles—associated with the weighted functional network. We leverage the detected topological information to define the homological scaffolds, a new set of objects designed to represent compactly the homological features of the correlation network and simultaneously make their homological properties amenable to networks theoretical methods. As a proof of principle, we apply these tools to compare resting-state functional brain activity in 15 healthy volunteers after intravenous infusion of placebo and psilocybin—the main psychoactive component of magic mushrooms. The results show that the homological structure of the brain's functional patterns undergoes a dramatic change post-psilocybin, characterized by the appearance of many transient structures of low stability and of a small number of persistent ones that are not observed in the case of placebo. PMID:25401177

  8. Homological scaffolds of brain functional networks.

    PubMed

    Petri, G; Expert, P; Turkheimer, F; Carhart-Harris, R; Nutt, D; Hellyer, P J; Vaccarino, F

    2014-12-06

    Networks, as efficient representations of complex systems, have appealed to scientists for a long time and now permeate many areas of science, including neuroimaging (Bullmore and Sporns 2009 Nat. Rev. Neurosci. 10, 186-198. (doi:10.1038/nrn2618)). Traditionally, the structure of complex networks has been studied through their statistical properties and metrics concerned with node and link properties, e.g. degree-distribution, node centrality and modularity. Here, we study the characteristics of functional brain networks at the mesoscopic level from a novel perspective that highlights the role of inhomogeneities in the fabric of functional connections. This can be done by focusing on the features of a set of topological objects-homological cycles-associated with the weighted functional network. We leverage the detected topological information to define the homological scaffolds, a new set of objects designed to represent compactly the homological features of the correlation network and simultaneously make their homological properties amenable to networks theoretical methods. As a proof of principle,we apply these tools to compare resting state functional brain activity in 15 healthy volunteers after intravenous infusion of placebo and psilocybin-the main psychoactive component of magic mushrooms. The results show that the homological structure of the brain's functional patterns undergoes a dramatic change post-psilocybin, characterized by the appearance of many transient structures of low stability and of a small number of persistent ones that are not observed in the case of placebo.

  9. Hyper(co)homology for exact left covariant functors and a homology theory for topological spaces

    NASA Astrophysics Data System (ADS)

    Sklyarenko, E. G.

    1995-06-01

    Contents Introduction §1. Strong cohomology of dual complexes §2. Hyperhomology §3. Examples §4. Typical limit relations for Steenrod-Sitnikov homology §5. The strong homology of topological spaces §6. On the special position held by singular theory Bibliography

  10. Flare build-up study: Homologous flares group - Interim report

    NASA Technical Reports Server (NTRS)

    Woodgate, B. E.

    1982-01-01

    When homologous flares are broadly defined as having footpoint structures in common, it is found that a majority of flares fall into homologous sets. Filament eruptions and mass ejection in members of an homologous flare set show that maintainance of the magnetic structure is not a necessary condition for homology.

  11. INVHOGEN: a database of homologous invertebrate genes.

    PubMed

    Paulsen, Ingo; von Haeseler, Arndt

    2006-01-01

    Classification of proteins into families of homologous sequences constitutes the basis of functional analysis or of evolutionary studies. Here we present INVertebrate HOmologous GENes (INVHOGEN), a database combining the available invertebrate protein genes from UniProt (consisting of Swiss-Prot and TrEMBL) into gene families. For each family INVHOGEN provides a multiple protein alignment, a maximum likelihood based phylogenetic tree and taxonomic information about the sequences. It is possible to download the corresponding GenBank flatfiles, the alignment and the tree in Newick format. Sequences and related information have been structured in an ACNUC database under a client/server architecture. Thus, complex selections can be performed. An external graphical tool (FamFetch) allows access to the data to evaluate homology relationships between genes and distinguish orthologous from paralogous sequences. Thus, INVHOGEN complements the well-known HOVERGEN database. The databank is available at http://www.bi.uni-duesseldorf.de/~invhogen/invhogen.html.

  12. Homologous recombination in plants: an antireview.

    PubMed

    Lieberman-Lazarovich, Michal; Levy, Avraham A

    2011-01-01

    Homologous recombination (HR) is a central cellular process involved in many aspects of genome maintenance such as DNA repair, replication, telomere maintenance, and meiotic chromosomal segregation. HR is highly conserved among eukaryotes, contributing to genome stability as well as to the generation of genetic diversity. It has been intensively studied, for almost a century, in plants and in other organisms. In this antireview, rather than reviewing existing knowledge, we wish to underline the many open questions in plant HR. We will discuss the following issues: how do we define homology and how the degree of homology affects HR? Are there any plant-specific HR qualities, how extensive is functional conservation and did HR proteins acquire new functions? How efficient is HR in plants and what are the cis and the trans factors that regulate it? Finally, we will give the prospects for enhancing the rates of gene targeting and meiotic HR for plant breeding purposes.

  13. Irradiated homologous costal cartilage for augmentation rhinoplasty

    SciTech Connect

    Lefkovits, G. )

    1990-10-01

    Although the ideal reconstructive material for augmentation rhinoplasty continues to challenge plastic surgeons, there exists no report in the literature that confines the use of irradiated homologous costal cartilage, first reported by Dingman and Grabb in 1961, to dorsal nasal augmentation. The purpose of this paper is to present a retrospective analysis of the author's experience using irradiated homologous costal cartilage in augmentation rhinoplasty. Twenty-seven dorsal nasal augmentations were performed in 24 patients between 16 and 49 years of age with a follow-up ranging from 1 to 27 months. Good-to-excellent results were achieved in 83.3% (20 of 24). Poor results requiring revision were found in 16.7% (4 of 24). Complication rates included 7.4% infection (2 of 27) and 14.8% warping (4 of 27). The resorption rate was zero. These results compare favorably with other forms of nasal augmentation. Advantages and disadvantages of irradiated homologous costal cartilage are discussed.

  14. Solar core homology, solar neutrinos and helioseismology

    SciTech Connect

    Bludman, S.A.; Kennedy, D.C.

    1995-12-31

    Precise numerical standard solar models (SSMs) now agree with one another and with helioseismological observations in the convective and outer radiative zones. Nevertheless these models obscure how luminosity, neutrino production and g-mode core helioseismology depend on such inputs as opacity and nuclear cross sections. Although the Sun is not homologous, its inner core by itself is chemically evolved and almost homologous, because of its compactness, radiative energy transport, and ppI-dominated luminosity production. We apply luminosity-fixed homology transformations to the core to estimate theoretical uncertainties in the SSM and to obtain a broad class of non-SSMs, parameterized by central temperature and density and purely radiative energy transport in the core. 25 refs., 3 figs., 3 tabs.

  15. Secretion of a chimeric T-cell receptor-immunoglobulin protein.

    PubMed Central

    Gascoigne, N R; Goodnow, C C; Dudzik, K I; Oi, V T; Davis, M M

    1987-01-01

    To produce sufficient quantities of soluble T-cell receptor protein for detailed biochemical and biophysical analyses we have explored the use of immunoglobulin--T-cell receptor gene fusions. In this report we describe a chimeric gene construct containing a T-cell receptor alpha-chain variable (V) domain and the constant (C) region coding sequences of an immunoglobulin gamma 2a molecule. Cells transfected with the chimeric gene synthesize a stable protein product that expresses immunoglobulin and T-cell receptor antigenic determinants as well as protein A binding sites. We show that the determinant recognized by the anticlonotypic antibody A2B4.2 resides on the V alpha domain of the T-cell receptor. The chimeric protein associates with a normal lambda light chain to form an apparently normal tetrameric (H2L2, where H = heavy and L = light) immunoglobulin molecule that is secreted. Also of potential significance is the fact that a T-cell receptor V beta gene in the same construct is neither assembled nor secreted with the lambda light chain, and when expressed with a C kappa region it does not assemble with the chimeric V alpha C gamma 2a protein mentioned above. This indicates that not all T-cell receptor V regions are similar enough to immunoglobulin V regions for them to be completely interchangeable. Images PMID:3472243

  16. Perceptual Asymmetry for Chimeric Stimuli in Children with Early Unilateral Brain Damage

    ERIC Educational Resources Information Center

    Bava, Sunita; Ballantyne, Angela O.; May, Susanne J.; Trauner, Doris A.

    2005-01-01

    The present study used a chimeric stimuli task to assess the magnitude of the left-hemispace bias in children with congenital unilateral brain damage (n=46) as compared to typically developing matched controls (n=46). As would be expected, controls exhibited a significant left-hemispace bias. In the presence of left hemisphere (LH) damage, the…

  17. Chimeric Ply187 endolysin kills Staphylococcus aureus more effectively than the parental enzyme.

    USDA-ARS?s Scientific Manuscript database

    Peptidoglycan hydrolases are an effective new source of antimicrobials. A chimeric fusion protein of the Ply187 endopeptidase domain and LysK SH3b cell wall binding domain is a potent agent against Staphylococcus aureus in three functional assays....

  18. Versatile bio-ink for covalent immobilization of chimeric avidin on sol-gel substrates.

    PubMed

    Heikkinen, Jarkko J; Kivimäki, Liisa; Määttä, Juha A E; Mäkelä, Inka; Hakalahti, Leena; Takkinen, Kristiina; Kulomaa, Markku S; Hytönen, Vesa P; Hormi, Osmo E O

    2011-10-15

    A bio-ink for covalent deposition of thermostable, high affinity biotin-binding chimeric avidin onto sol-gel substrates was developed. The bio-ink was prepared from heterobifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide which was first reacted either with 3-aminopropyltriethoxysilane or 3-aminopropyldimethylethoxysilane to form silane linkers 6-maleimide-N-(3-(triethoxysilyl)propyl)hexanamide or -(ethoxydimethylsilyl)propyl)-hexanamide. C-terminal cysteine genetically engineered to chimeric avidin was reacted with the maleimide group of silane linker in methanol/PBS solution to form a suspension, which was printed on sol-gel modified PMMA film. Different concentrations of chimeric avidin and ratios between silane linkers were tested to find the best properties for the bio-ink to enable gravure or inkjet printing. Bio-ink prepared from 3-aminopropyltriethoxysilane was found to provide the highest amount of active immobilized chimeric avidin. The developed bio-ink was shown to be valuable for automated fabrication of avidin-functionalized polymer films. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms

    NASA Technical Reports Server (NTRS)

    Kopczynski, E. D.; Bateson, M. M.; Ward, D. M.

    1994-01-01

    When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered. They were revealed as chimeras of SSU rRNA genes of uncultivated species through separate phylogenetic analysis of short sequence domains.

  20. Trypanosoma cruzi Differentiates and Multiplies within Chimeric Parasitophorous Vacuoles in Macrophages Coinfected with Leishmania amazonensis.

    PubMed

    Pessoa, Carina Carraro; Ferreira, Éden Ramalho; Bayer-Santos, Ethel; Rabinovitch, Michel; Mortara, Renato Arruda; Real, Fernando

    2016-05-01

    The trypanosomatids Leishmania amazonensis and Trypanosoma cruzi are excellent models for the study of the cell biology of intracellular protozoan infections. After their uptake by mammalian cells, the parasitic protozoan flagellates L. amazonensis and T. cruzi lodge within acidified parasitophorous vacuoles (PVs). However, whereas L. amazonensis develops in spacious, phagolysosome-like PVs that may enclose numerous parasites, T. cruzi is transiently hosted within smaller vacuoles from which it soon escapes to the host cell cytosol. To investigate if parasite-specific vacuoles are required for the survival and differentiation of T. cruzi, we constructed chimeric vacuoles by infection of L. amazonensis amastigote-infected macrophages with T. cruzi epimastigotes (EPIs) or metacyclic trypomastigotes (MTs). These chimeric vacuoles, easily observed by microscopy, allowed the entry and fate of T. cruzi in L. amazonensis PVs to be dynamically recorded by multidimensional imaging of coinfected cells. We found that although T. cruzi EPIs remained motile and conserved their morphology in chimeric vacuoles, T. cruzi MTs differentiated into amastigote-like forms capable of multiplying. These results demonstrate that the large adaptive vacuoles of L. amazonensis are permissive to T. cruzi survival and differentiation and that noninfective EPIs are spared from destruction within the chimeric PVs. We conclude that T. cruzi differentiation can take place in Leishmania-containing vacuoles, suggesting this occurs prior to their escape into the host cell cytosol.

  1. The Evolution and Analysis of the Functional Domains of the Chimeric Proteins that Initiate Pyrimidine Biosynthesis

    DTIC Science & Technology

    1989-10-15

    proteins in eubacteria , but are consolidated in a single 243 kDa chimeric plypeptide in mrnmals and other higher eukaryotes. We have shown previously that...and regulatory properties, have been identified in eubacteria . E. coli aspartate transcarbamylase, a well characterized class B enzyme, Is a

  2. Growth and long-term somatic and germline chimerism following fusion of juvenile Botryllus schlosseri.

    PubMed

    Carpenter, Meredith A; Powell, John H; Ishizuka, Katherine J; Palmeri, Karla J; Rendulic, Snjezana; De Tomaso, Anthony W

    2011-02-01

    The colonial ascidian Botryllus schlosseri undergoes a histocompatibility reaction that can result in vascular fusion of distinct genotypes, creating a chimera. Chimerism has both potential benefits, such as an immediate increase in size that may enhance growth rates, and costs. For the latter, the presence of multiple genotypes in a chimera can lead to competition between genetically distinct stem cell lineages, resulting in complete replacement of somatic and germline tissues by a single genotype. Although fusion can occur at any point after metamorphosis, previous studies have focused on chimeras created from sexually mature adults, where no benefit to chimerism has been documented. Here we focus on the costs and benefits of fusion between juveniles, characterizing growth rates and patterns of somatic and germline chimerism after natural and controlled fusion events. We also compared outcomes between low- and high-density growth conditions, the latter more likely representative of what occurs in natural populations. We found that growth rates were density-dependent, and that only chimeras grew under high-density conditions. We also observed a positional component to a post-fusion event called resorption, indicating that extrinsic factors were important in this process. Patterns of germline and somatic chimerism and dominance in chimeras made from fused juveniles were equivalent to those after fusion of sexually mature adults, and there were no age-related differences in these processes. Finally, by using genetic markers that could retrospectively assign genotypes, we also found that the majority of individual testes in a chimera were clonally derived.

  3. Design, synthesis and bioactivity of catechin/epicatechin and 2-azetidinone derived chimeric molecules.

    PubMed

    Roy, Basab; Chakraborty, Arindam; Ghosh, Sudip K; Basak, Amit

    2009-12-15

    A new class of chimeric molecules have been developed. These are based on polyphenols like catechin and epicatechin and monocyclic beta-lactams. The two units are joined via a triazole linker using the 'Click Chemistry' conditions. The compounds showed good to weak antibacterial activity against Escherichia coli as well as moderate inhibition of RNase A.

  4. Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes

    PubMed Central

    Fischbach, Michael A.; Lai, Jonathan R.; Roche, Eric D.; Walsh, Christopher T.; Liu, David R.

    2007-01-01

    Nonribosomal peptides (NRPs) are produced by NRP synthetase (NRPS) enzymes that function as molecular assembly lines. The modular architecture of NRPSs suggests that a domain responsible for activating a building block could be replaced with a domain from a foreign NRPS to create a chimeric assembly line that produces a new variant of a natural NRP. However, such chimeric NRPS modules are often heavily impaired, impeding efforts to create novel NRP variants by swapping domains from different modules or organisms. Here we show that impaired chimeric NRPSs can be functionally restored by directed evolution. Using rounds of mutagenesis coupled with in vivo screens for NRP production, we rapidly isolated variants of two different chimeric NRPSs with ≈10-fold improvements in enzyme activity and product yield, including one that produces new derivatives of the potent NRP/polyketide antibiotic andrimid. Because functional restoration in these examples required only modest library sizes (103 to 104 clones) and three or fewer rounds of screening, our approach may be widely applicable even for NRPSs from genetically challenging hosts. PMID:17620609

  5. A Chimeric Pneumovirus Fusion Protein Carrying Neutralizing Epitopes of Both MPV and RSV

    PubMed Central

    Wen, Xiaolin; Pickens, Jennifer; Mousa, Jarrod J.; Leser, George P.; Lamb, Robert A.; Crowe, James E.; Jardetzky, Theodore S.

    2016-01-01

    Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are paramyxoviruses that are responsible for substantial human health burden, particularly in children and the elderly. The fusion (F) glycoproteins are major targets of the neutralizing antibody response and studies have mapped dominant antigenic sites in F. Here we grafted a major neutralizing site of RSV F, recognized by the prophylactic monoclonal antibody palivizumab, onto HMPV F, generating a chimeric protein displaying epitopes of both viruses. We demonstrate that the resulting chimeric protein (RPM-1) is recognized by both anti-RSV and anti-HMPV F neutralizing antibodies indicating that it can be used to map the epitope specificity of antibodies raised against both viruses. Mice immunized with the RPM-1 chimeric antigen generate robust neutralizing antibody responses to MPV but weak or no cross-reactive recognition of RSV F, suggesting that grafting of the single palivizumab epitope stimulates a comparatively limited antibody response. The RPM-1 protein provides a new tool for characterizing the immune responses resulting from RSV and HMPV infections and provides insights into the requirements for developing a chimeric subunit vaccine that could induce robust and balanced immunity to both virus infections. PMID:27224013

  6. Trypanosoma cruzi Differentiates and Multiplies within Chimeric Parasitophorous Vacuoles in Macrophages Coinfected with Leishmania amazonensis

    PubMed Central

    Pessoa, Carina Carraro; Ferreira, Éden Ramalho; Bayer-Santos, Ethel; Rabinovitch, Michel; Mortara, Renato Arruda

    2016-01-01

    The trypanosomatids Leishmania amazonensis and Trypanosoma cruzi are excellent models for the study of the cell biology of intracellular protozoan infections. After their uptake by mammalian cells, the parasitic protozoan flagellates L. amazonensis and T. cruzi lodge within acidified parasitophorous vacuoles (PVs). However, whereas L. amazonensis develops in spacious, phagolysosome-like PVs that may enclose numerous parasites, T. cruzi is transiently hosted within smaller vacuoles from which it soon escapes to the host cell cytosol. To investigate if parasite-specific vacuoles are required for the survival and differentiation of T. cruzi, we constructed chimeric vacuoles by infection of L. amazonensis amastigote-infected macrophages with T. cruzi epimastigotes (EPIs) or metacyclic trypomastigotes (MTs). These chimeric vacuoles, easily observed by microscopy, allowed the entry and fate of T. cruzi in L. amazonensis PVs to be dynamically recorded by multidimensional imaging of coinfected cells. We found that although T. cruzi EPIs remained motile and conserved their morphology in chimeric vacuoles, T. cruzi MTs differentiated into amastigote-like forms capable of multiplying. These results demonstrate that the large adaptive vacuoles of L. amazonensis are permissive to T. cruzi survival and differentiation and that noninfective EPIs are spared from destruction within the chimeric PVs. We conclude that T. cruzi differentiation can take place in Leishmania-containing vacuoles, suggesting this occurs prior to their escape into the host cell cytosol. PMID:26975994

  7. Perceptual Asymmetry for Chimeric Stimuli in Children with Early Unilateral Brain Damage

    ERIC Educational Resources Information Center

    Bava, Sunita; Ballantyne, Angela O.; May, Susanne J.; Trauner, Doris A.

    2005-01-01

    The present study used a chimeric stimuli task to assess the magnitude of the left-hemispace bias in children with congenital unilateral brain damage (n=46) as compared to typically developing matched controls (n=46). As would be expected, controls exhibited a significant left-hemispace bias. In the presence of left hemisphere (LH) damage, the…

  8. Alloreactive Regulatory T Cells Allow the Generation of Mixed Chimerism and Transplant Tolerance.

    PubMed

    Ruiz, Paulina; Maldonado, Paula; Hidalgo, Yessia; Sauma, Daniela; Rosemblatt, Mario; Bono, Maria Rosa

    2015-01-01

    The induction of donor-specific transplant tolerance is one of the main goals of modern immunology. Establishment of a mixed chimerism state in the transplant recipient has proven to be a suitable strategy for the induction of long-term allograft tolerance; however, current experimental recipient preconditioning protocols have many side effects, and are not feasible for use in future therapies. In order to improve the current mixed chimerism induction protocols, we developed a non-myeloablative bone-marrow transplant (NM-BMT) protocol using retinoic acid (RA)-induced alloantigen-specific Tregs, clinically available immunosuppressive drugs, and lower doses of irradiation. We demonstrate that RA-induced alloantigen-specific Tregs in addition to a NM-BMT protocol generates stable mixed chimerism and induces tolerance to allogeneic secondary skin allografts in mice. Therefore, the establishment of mixed chimerism through the use of donor-specific Tregs rather than non-specific immunosuppression could have a potential use in organ transplantation.

  9. Alloreactive Regulatory T Cells Allow the Generation of Mixed Chimerism and Transplant Tolerance

    PubMed Central

    Ruiz, Paulina; Maldonado, Paula; Hidalgo, Yessia; Sauma, Daniela; Rosemblatt, Mario; Bono, Maria Rosa

    2015-01-01

    The induction of donor-specific transplant tolerance is one of the main goals of modern immunology. Establishment of a mixed chimerism state in the transplant recipient has proven to be a suitable strategy for the induction of long-term allograft tolerance; however, current experimental recipient preconditioning protocols have many side effects, and are not feasible for use in future therapies. In order to improve the current mixed chimerism induction protocols, we developed a non-myeloablative bone-marrow transplant (NM-BMT) protocol using retinoic acid (RA)-induced alloantigen-specific Tregs, clinically available immunosuppressive drugs, and lower doses of irradiation. We demonstrate that RA-induced alloantigen-specific Tregs in addition to a NM-BMT protocol generates stable mixed chimerism and induces tolerance to allogeneic secondary skin allografts in mice. Therefore, the establishment of mixed chimerism through the use of donor-specific Tregs rather than non-specific immunosuppression could have a potential use in organ transplantation. PMID:26635810

  10. Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms

    NASA Technical Reports Server (NTRS)

    Kopczynski, E. D.; Bateson, M. M.; Ward, D. M.

    1994-01-01

    When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered. They were revealed as chimeras of SSU rRNA genes of uncultivated species through separate phylogenetic analysis of short sequence domains.

  11. [Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector].

    PubMed

    Pan, X-X; Zhao, B-X; Teng, Y-M; Xia, W-Y; Wang, J; Li, X-F; Liao, G-Y; Yang, С; Chen, Y-D

    2016-01-01

    Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.

  12. In Silico Design of a Chimeric Protein Containing Antigenic Fragments of Helicobacter pylori; A Bioinformatic Approach

    PubMed Central

    Mohammad, Nazanin; Karsabet, Mehrnaz Taghipour; Amani, Jafar; Ardjmand, Abolfazl; Zadeh, Mohsen Razavi; Gholi, Mohammad Khalifeh; Saffari, Mahmood; Ghasemi, Amir

    2016-01-01

    Helicobacter pylori is a global health problem which has encouraged scientists to find new ways to diagnose, immunize and eradicate the H. pylori infection. In silico studies are a promising approach to design new chimeric antigen having the immunogenic potential of several antigens. In order to obtain such benefit in H. pylori vaccine study, a chimeric gene containing four fragments of FliD sequence (1-600 bp), UreB (327-334 bp),VacA (744-805 bp) and CagL(51-100 bp) which have a high density of B- and T-cell epitopes was designed. The secondary and tertiary structures of the chimeric protein and other properties such as stability, solubility and antigenicity were analyzed. The in silico results showed that after optimizing for the purpose of expression in Escherichia coli BL21, the solubility and antigenicity of the construct fragments were highly retained. Most regions of the chimeric protein were found to have a high antigenic propensity and surface accessibility. These results would be useful in animal model application and accounted for the development of an epitope-based vaccine against the H. pylori. PMID:27335622

  13. Intravitreal injection of a chimeric phage endolysin Ply187 protects mice from Staphylococcus aureus endophthalmitis

    USDA-ARS?s Scientific Manuscript database

    Objectives: The treatment of endophthalmitis is becoming very challenging due to the emergence of multidrug-resistant bacteria. Hence, the development of novel therapeutic alternatives for ocular use is essential. Here, we evaluated the therapeutic potential of Ply187AN-KSH3b, a chimeric phage endol...

  14. Multipaddled Anterolateral Thigh Chimeric Flap for Reconstruction of Complex Defects in Head and Neck

    PubMed Central

    Li, Ning; Liu, Wen; Su, Tong; Chen, Xinqun; Zheng, Lian; Jian, Xinchun

    2014-01-01

    The anterolateral thigh flap has been the workhouse flap for coverage of soft-tissue defects in head and neck for decades. However, the reconstruction of multiple and complex soft-tissue defects in head and neck with multipaddled anterolateral thigh chimeric flaps is still a challenge for reconstructive surgeries. Here, a clinical series of 12 cases is reported in which multipaddled anterolateral thigh chimeric flaps were used for complex soft-tissue defects with several separately anatomic locations in head and neck. Of the 12 cases, 7 patients presented with trismus were diagnosed as advanced buccal cancer with oral submucous fibrosis, 2 tongue cancer cases were found accompanied with multiple oral mucosa lesions or buccal cancer, and 3 were hypopharyngeal cancer with anterior neck skin invaded. All soft-tissue defects were reconstructed by multipaddled anterolateral thigh chimeric flaps, including 9 tripaddled anterolateral thigh flaps and 3 bipaddled flaps. The mean length of skin paddle was 19.2 (range: 14–23) cm and the mean width was 4.9 (range: 2.5–7) cm. All flaps survived and all donor sites were closed primarily. After a mean follow-up time of 9.1 months, there were no problems with the donor or recipient sites. This study supports that the multipaddled anterolateral thigh chimeric flap is a reliable and good alternative for complex and multiple soft-tissue defects of the head and neck. PMID:25180680

  15. Evidence for Transcript Networks Composed of Chimeric RNAs in Human Cells

    PubMed Central

    Borel, Christelle; Mudge, Jonathan M.; Howald, Cédric; Foissac, Sylvain; Ucla, Catherine; Chrast, Jacqueline; Ribeca, Paolo; Martin, David; Murray, Ryan R.; Yang, Xinping; Ghamsari, Lila; Lin, Chenwei; Bell, Ian; Dumais, Erica; Drenkow, Jorg; Tress, Michael L.; Gelpí, Josep Lluís; Orozco, Modesto; Valencia, Alfonso; van Berkum, Nynke L.; Lajoie, Bryan R.; Vidal, Marc; Stamatoyannopoulos, John; Batut, Philippe; Dobin, Alex; Harrow, Jennifer; Hubbard, Tim; Dekker, Job; Frankish, Adam; Salehi-Ashtiani, Kourosh; Reymond, Alexandre; Antonarakis, Stylianos E.; Guigó, Roderic; Gingeras, Thomas R.

    2012-01-01

    The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5′ and 3′ transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network. PMID:22238572

  16. Mixed chimerism in haemoglobinopathies: from risk of graft rejection to immune tolerance.

    PubMed

    Andreani, M; Testi, M; Lucarelli, G

    2014-03-01

    Mixed chimerism (MC), the simultaneous presence of both host- and donor-derived cells in the recipient, is observed in a large proportion of patients after haematopoietic stem cell transplant (HSCT) to treat haemoglobinopathies. Detected early after transplantation, MC often moves towards complete chimerism, although sometimes it may evolve into graft rejection, especially if the proportion of donor cells is very low. However, some patients develop stable MC, defined as persistent when donor- and host-derived cells coexist for periods longer than 2 years after HSCT. Patients with persistent mixed chimerism (PMC) do not require additional red blood cell support and, regardless of the presence in some cases of an extremely low percentage of donor-derived nucleated cells in the bone marrow, their condition is clinically controlled by an incomplete but functional graft, as they express a two- to fivefold enrichment of donor-derived mature erythrocytes in the peripheral blood. These findings have tremendous implications not only in the context of allogeneic HSCT but also in the design of gene therapy trials based on the autologous transplantation of genetically modified CD34+ cells. Recent studies have shown that durable allograft tolerance has been achieved by induction of haematopoietic chimerism in clinical kidney transplantation, showing the involvement of regulatory T cells. Similarly, it has been shown that the regulatory T cells play a pivotal role in promoting and maintaining immune tolerance in patients that develop a status of PMC after HSCT for Thalassemia.

  17. Chimeric antigen receptor-engineered T cells for liver cancers, progress and obstacles.

    PubMed

    Li, Keyu; Lan, Yaliang; Wang, Jiabei; Liu, Lianxin

    2017-03-01

    Chimeric antigen receptor-engineered T cells therapy has become the hottest topic of immunotherapy, as its great successes achieved in treating refractory hematological malignancies. These successes also paved the road to novel strategies of treating various solid tumors including liver cancer. Many specific proteins can be expressed aberrantly in liver cancers; therefore, a series of experimental and clinical researches exploring chimeric antigen receptor-engineered T cells and liver cancer are in progress, acquiring obvious antitumor effect and revealing its feasibility in treating liver cancer. However, lots of challenges and obstacles are emerging simultaneously, such as low infiltration, side effects, safety of chimeric antigen receptor-engineered T cells, and limited data of studies or clinical trials. Researchers have been working out many innovative ways to directly stroke these obstacles, theoretically or practically. This review focuses more on the progress and obstacles from chimeric antigen receptor-engineered T cells therapy to treat liver cancer, summarizing new breakthroughs in shooting those obstacles, meanwhile, hoping to provide enlightenment to this promising immunotherapeutic method.

  18. Engineered Chimeric Peptides as Antimicrobial Surface Coating Agents toward Infection-Free Implants

    PubMed Central

    Yazici, Hilal; O'Neill, Mary B.; Kacar, Turgay; Wilson, Brandon R.; Oren, E. Emre; Sarikaya, Mehmet; Tamerler, Candan

    2016-01-01

    Prevention of bacterial colonization and consequent biofilm formation remains a major challenge in implantable medical devices. Implant-associated infections are not only a major cause of implant failures but also their conventional treatment with antibiotics brings further complications due to the escalation in multidrug resistance to a variety of bacterial species. Owing to their unique properties, antimicrobial peptides (AMPs) have gained significant attention as effective agents to combat colonization of microorganisms. These peptides have been shown to exhibit a wide spectrum of activities with specificity to a target cell while having a low tendency for developing bacterial resistance. Engineering biomaterial surfaces that feature AMP properties, therefore, offer a promising approach to prevent implant infections. Here, we engineered a chimeric peptide with bifunctionality that both forms a robust solid-surface coating while presenting antimicrobial property. The individual domains of the chimeric peptides were evaluated for their solid-binding kinetics to titanium substrate as well as for their antimicrobial properties in solution. The antimicrobial efficacy of the chimeric peptide on the implant material was evaluated in vitro against infection by a variety of bacteria, including Streptococcus mutans, Staphylococcus. epidermidis, and Escherichia coli, which are commonly found in oral and orthopedic implant related surgeries. Our results demonstrate significant improvement in reducing bacterial colonization onto titanium surfaces below the detectable limit. Engineered chimeric peptides with freely displayed antimicrobial domains could be a potential solution for developing infection-free surfaces by engineering implant interfaces with highly reduced bacterial colonization property. PMID:26795060

  19. Engineered Chimeric Peptides as Antimicrobial Surface Coating Agents toward Infection-Free Implants.

    PubMed

    Yazici, Hilal; O'Neill, Mary B; Kacar, Turgay; Wilson, Brandon R; Oren, E Emre; Sarikaya, Mehmet; Tamerler, Candan

    2016-03-02

    Prevention of bacterial colonization and consequent biofilm formation remains a major challenge in implantable medical devices. Implant-associated infections are not only a major cause of implant failures but also their conventional treatment with antibiotics brings further complications due to the escalation in multidrug resistance to a variety of bacterial species. Owing to their unique properties, antimicrobial peptides (AMPs) have gained significant attention as effective agents to combat colonization of microorganisms. These peptides have been shown to exhibit a wide spectrum of activities with specificity to a target cell while having a low tendency for developing bacterial resistance. Engineering biomaterial surfaces that feature AMP properties, therefore, offer a promising approach to prevent implant infections. Here, we engineered a chimeric peptide with bifunctionality that both forms a robust solid-surface coating while presenting antimicrobial property. The individual domains of the chimeric peptides were evaluated for their solid-binding kinetics to titanium substrate as well as for their antimicrobial properties in solution. The antimicrobial efficacy of the chimeric peptide on the implant material was evaluated in vitro against infection by a variety of bacteria, including Streptococcus mutans, Staphylococcus. epidermidis, and Escherichia coli, which are commonly found in oral and orthopedic implant related surgeries. Our results demonstrate significant improvement in reducing bacterial colonization onto titanium surfaces below the detectable limit. Engineered chimeric peptides with freely displayed antimicrobial domains could be a potential solution for developing infection-free surfaces by engineering implant interfaces with highly reduced bacterial colonization property.

  20. Chimeric RNase H-competent oligonucleotides directed to the HIV-1 Rev response element.

    PubMed

    Prater, Chrissy E; Saleh, Anthony D; Wear, Maggie P; Miller, Paul S

    2007-08-15

    Chimeric oligo-2'-O-methylribonucleotides containing centrally located patches of contiguous 2'-deoxyribonucleotides and terminating in a nuclease resistant 3'-methylphosphonate internucleotide linkage were prepared. The oligonucleotides were targeted to the 3'-side of HIV Rev response element (RRE) stem-loop IIB RNA, which is adjacent to the high affinity Rev protein binding site and is critical to virus function. Thermal denaturation experiments showed that chimeric oligonucleotides form very stable duplexes with a complementary single-stranded RNA, and gel electrophoretic mobility shift assays (EMSA) showed that they bind with high affinity and specificity to RRE stem-loop II RNA (K(D) approximately 200 nM). The chimeric oligonucleotides promote RNase H-mediated hydrolysis of RRE stem-loop II RNA and have half-lives exceeding 24h when incubated in cell culture medium containing 10% fetal calf serum. One of the chimeric oligonucleotides inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) approximately 60% at a concentration of 300 nM in HEK 293T cells co-transfected with p-RRE/CAT and p-Rev mammalian expression vectors.

  1. A mathematical model for the rational design of chimeric ligands in selective drug therapies.

    PubMed

    Doldán-Martelli, V; Guantes, R; Míguez, D G

    2013-02-13

    Chimeric drugs with selective potential toward specific cell types constitute one of the most promising forefronts of modern Pharmacology. We present a mathematical model to test and optimize these synthetic constructs, as an alternative to conventional empirical design. We take as a case study a chimeric construct composed of epidermal growth factor (EGF) linked to different mutants of interferon (IFN). Our model quantitatively reproduces all the experimental results, illustrating how chimeras using mutants of IFN with reduced affinity exhibit enhanced selectivity against cell overexpressing EGF receptor. We also investigate how chimeric selectivity can be improved based on the balance between affinity rates, receptor abundance, activity of ligand subunits, and linker length between subunits. The simplicity and generality of the model facilitate a straightforward application to other chimeric constructs, providing a quantitative systematic design and optimization of these selective drugs against certain cell-based diseases, such as Alzheimer's and cancer.CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e26; doi:10.1038/psp.2013.2; advance online publication 13 February 2013.

  2. The perforator-based conjoint (chimeric) medial Sural(MEDIAL GASTROCNEMIUS) free flap.

    PubMed

    Sano, Kazufumi; Hallock, Geoffrey G; Hamazaki, Masahiro; Daicyo, Yoshihiro

    2004-12-01

    The prototypical conjoint or so-called "chimeric" free flap heretofore has been composed of several large independent flaps, each supplied by a separate major branch, that ultimately arise from a common source vessel. The perforator-based type of chimeric flap is a relatively new concept, usually involving multiple muscle perforator flaps each based on a solitary musculocutaneous perforator, but still arising from the same "mother" vessel. This principle of split cutaneous perforator flaps has been now successfully adapted to the medial suralMEDIAL GASTROCNEMIUS perforator free flap on 2 separate occasions. As a chimeric flap, there was greater flexibility in insetting, and overall flap width may be larger but still narrow enough to allow primary donor site closure; and yet, by definition, only a single recipient site was needed for any microanastomoses. This is further proof that the perforator-based chimeric free flap may be an option for any muscle perforator flap donor site, so that potential donor territories for conjoint flaps have become virtually unlimited.

  3. Low levels of allogeneic but not syngeneic hematopoietic chimerism reverse autoimmune insulitis in prediabetic NOD mice.

    PubMed

    Kaminitz, Ayelet; Mizrahi, Keren; Yaniv, Isaac; Farkas, Daniel L; Stein, Jerry; Askenasy, Nadir

    2009-09-01

    The relative efficiencies of allogeneic and syngeneic bone marrow transplantation and the threshold levels of donor chimerism required to control autoimmune insulitis were evaluated in prediabetic NOD mice. Male and female NOD mice were conditioned by radiation and grafted with bone marrow cells from allogeneic and syngeneic sex-mismatched donors. Establishment of full allogeneic chimerism in peripheral blood reversed insulitis and restored glucose tolerance despite persistence of residual host immune cells. By contrast, sublethal total body irradiation (with or without syngeneic transplant) reduced the incidence and delayed the onset of diabetes. The latter pattern was also seen in mice that rejected the bone marrow allografts. Low levels of stable allogeneic hematopoietic chimerism (>1%) were sufficient to prevent the evolution of diabetes following allogeneic transplantation. The data indicate that immunomodulation attained at low levels of allogeneic, but not syngeneic, hematopoietic chimerism is effective in resolution of islet inflammation at even relatively late stages in the evolution of the prediabetic state in a preclinical model. However, our data question the efficacy and rationale behind syngeneic (autologous-like) immuno-hematopoietic reconstitution in type 1 diabetes.

  4. Blood ties: chimerism can mask twin discordance in high-throughput sequencing.

    PubMed

    Erlich, Yaniv

    2011-04-01

    Twin studies have long provided a means to separate the contributions of genetic and environmental factors. A recent pioneering report by Baranzini et al. presented an analysis of the complete genomes and epigenomes of a monozygotic (MZ) twin pair discordant for multiple sclerosis. This failed to find any difference between the twins, raising doubts regarding the value of whole-genome twin studies for defining disease susceptibility alleles. However, the study was carried out with DNA extracted from blood. In many cases, the hematopoietic lineages of MZ twins are chimeric due to twin-to-twin exchange of hematopoietic stem cells during embryogenesis. We therefore wondered how chimerism might impact the ability to identify genetic differences. We inferred the blood chimerism rates and profiles of more than 30 discordant twin cases from a wide variety of medical conditions. We found that the genotype compositions of the twins were highly similar. We then benchmarked the performance of SNP callers to detect discordant variations using high-throughput sequencing data. Our analysis revealed that chimerism patterns, well within the range normally observed in MZ twins, greatly reduce the sensitivity of SNP calls. This raises questions regarding any conclusions of genomic homogeneity that might be drawn from studies of blood-derived twin DNA.

  5. Chimeric peptide beacons: a direct polypeptide analog of DNA molecular beacons†

    PubMed Central

    Oh, Kenneth J.; Cash, Kevin J.; Lubin, Arica A.

    2009-01-01

    We have developed a new biosensor architecture, which is comprised of a polypeptide–peptide nucleic acid tri-block copolymer and which we have termed chimeric peptide beacons (CPB), that generates an optical output via a mechanism analogous to that employed in DNA-based molecular beacons. PMID:18361352

  6. CRES-T, an effective gene silencing system utilizing chimeric repressors.

    PubMed

    Mitsuda, Nobutaka; Matsui, Kyoko; Ikeda, Miho; Nakata, Masaru; Oshima, Yoshimi; Nagatoshi, Yukari; Ohme-Takagi, Masaru

    2011-01-01

    Chimeric REpressor gene Silencing Technology (CRES-T) is a useful tool for functional analysis of plant transcription factors. In this system, a chimeric repressor that is produced by fusion of a transcription factor to the plant-specific EAR-motif repression domain (SRDX) suppresses target genes of a transcription factor dominantly over the activity of endogenous and functionally redundant transcription factors. As a result, the transgenic plants that express a chimeric repressor exhibit phenotypes similar to loss-of-function of the alleles of the gene encoding the transcription factor. This system is simple and effective and can be used as a powerful tool not only for functional analysis of redundant transcription factors but also for the manipulation of plant traits by active suppression of the gene expression. Strategies for construction of the chimeric repressors and their expression in transgenic plants are described. Transient effector-reporter assays for functional analysis of transcription factors and detection of protein-protein interactions using the trans-repressive activity of SRDX repression domain are also described.

  7. Enhanced antibody-dependent cellular phagocytosis by chimeric monoclonal antibodies with tandemly repeated Fc domains.

    PubMed

    Nagashima, Hiroaki; Ootsubo, Michiko; Fukazawa, Mizuki; Motoi, Sotaro; Konakahara, Shu; Masuho, Yasuhiko

    2011-04-01

    We previously reported that chimeric monoclonal antibodies (mAbs) with tandemly repeated Fc domains, which were developed by introducing tandem repeats of Fc domains downstream of 2 Fab domains, augmented binding avidities for all Fcγ receptors, resulting in enhanced antibody (Ab)-dependent cellular cytotoxicity. Here we investigated regarding Ab-dependent cellular phagocytosis (ADCP) mediated by these chimeric mAbs, which is considered one of the most important mechanisms that kills tumor cells, using two-color flow cytometric methods. ADCP mediated by T3-Ab, a chimeric mAb with 3 tandemly repeated Fc domains, was 5 times more potent than that by native anti-CD20 M-Ab (M-Ab hereafter). Furthermore, T3-Ab-mediated ADCP was resistant to competitive inhibition by intravenous Ig (IVIG), although M-Ab-mediated ADCP decreased in the presence of IVIG. An Fcγ receptor-blocking study demonstrated that T3-Ab mediated ADCP via both FcγRIA and FcγRIIA, whereas M-Ab mediated ADCP exclusively via FcγRIA. These results suggest that chimeric mAbs with tandemly repeated Fc domains enhance ADCP as well as ADCC, and that Fc multimerization may significantly enhance the efficacy of therapeutic Abs.

  8. Selective targeting of human cells by a chimeric adenovirus vector containing a modified fiber protein.

    PubMed Central

    Stevenson, S C; Rollence, M; Marshall-Neff, J; McClelland, A

    1997-01-01

    The adenovirus fiber protein is responsible for attachment of the virion to unidentified cell surface receptors. There are at least two distinct adenovirus fiber receptors which interact with the group B (Ad3) and group C (Ad5) adenoviruses. We have previously shown by using expressed adenovirus fiber proteins that it is possible to change the specificity of the fiber protein by exchanging the head domain with another serotype which recognizes a different receptor (S. C. Stevenson et al., J. Virol. 69:2850-2857, 1995). A chimeric fiber cDNA containing the Ad3 fiber head domain fused to the Ad5 fiber tail and shaft was incorporated into the genome of an adenovirus vector with E1 and E3 deleted encoding beta-galactosidase to generate Av9LacZ4, an adenovirus particle which contains a chimeric fiber protein. Western blot analysis of the chimeric fiber vector confirmed expression of the chimeric fiber protein and its association with the adenovirus capsid. Transduction experiments with fiber protein competitors demonstrated the altered receptor tropism of the chimeric fiber vector compared to that of the parental Av1LacZ4 vector. Transduction of a panel of human cell lines with the chimeric and parental vectors provided evidence for a different cellular distribution of the Ad5 and Ad3 receptors. Three cell lines (THP-1, MRC-5, and FaDu) were more efficiently transduced by the vector containing the Ad3 fiber head than by the Ad5 fiber vector. In contrast, human coronary artery endothelial cells were transduced more readily with the vector containing the Ad5 fiber than with the chimeric fiber vector. HeLa and human umbilical vein endothelial cells were transduced at equivalent levels compared with human diploid fibroblasts, which were refractory to transduction with both vectors. These results provide evidence for the differential expression of the Ad5 and Ad3 receptors on human cell lines derived from clinically relevant target tissues. Furthermore, we show that exchange

  9. The rapid generation of chimerical genes expanding protein diversity in zebrafish.

    PubMed

    Fu, Beide; Chen, Ming; Zou, Ming; Long, Manyuan; He, Shunping

    2010-11-24

    Variation of gene number among species indicates that there is a general process of new gene origination. One of the major mechanism providing raw materials for the origin of new genes is gene duplication. Retroposition, as a special type of gene duplication- the RNA-based duplication, has been found to play an important role in new gene evolution in mammals and plants, but little is known about the process in the teleostei genome. Here we screened the zebrafish genome for identification of retrocopies and new chimerical retrogenes and investigated their origination and evolution. We identified 652 retrocopies, of which 440 are intact retrogenes and 212 are pseudogenes. Retrocopies have long been considered evolutionary dead ends without functional significance due to the presumption that retrocopies lack the regulatory element needed for expression. However, 437 transcribed retrocopies were identified from all of the retrocopies. This discovery combined with the substitution analysis suggested that the majority of all retrocopies are subject to negative selection, indicating that most of the retrocopies may be functional retrogenes. Moreover, we found that 95 chimerical retrogenes had recruited new sequences from neighboring genomic regions that formed de novo splice sites, thus generating new intron-containing chimeric genes. Based on our analysis of 38 pairs of orthologs between Cyprinus carpio and Danio rerio, we found that the synonymous substitution rate of zebrafish genes is 4.13×10⁻⁹ substitution per silent site per year. We also found 10 chimerical retrogenes that were created in the last 10 million years, which is 7.14 times the rate of 0.14 chimerical retrogenes per million years in the primate lineage toward human and 6.25 times the rate of 0.16 chimerical genes per million years in Drosophila. This is among the most rapid rates of generation of chimerical genes, just next to the rice. There is compelling evidence that much of the extensive

  10. A Bacteriophage-Related Chimeric Marine Virus Infecting Abalone

    PubMed Central

    Zhuang, Jun; Cai, Guiqin; Lin, Qiying; Wu, Zujian; Xie, Lianhui

    2010-01-01

    Marine viruses shape microbial communities with the most genetic diversity in the sea by multiple genetic exchanges and infect multiple marine organisms. Here we provide proof from experimental infection that abalone shriveling syndrome-associated virus (AbSV) can cause abalone shriveling syndrome. This malady produces histological necrosis and abnormally modified macromolecules (hemocyanin and ferritin). The AbSV genome is a 34.952-kilobase circular double-stranded DNA, containing putative genes with similarity to bacteriophages, eukaryotic viruses, bacteria and endosymbionts. Of the 28 predicted open reading frames (ORFs), eight ORF-encoded proteins have identifiable functional homologues. The 4 ORF products correspond to a predicted terminase large subunit and an endonuclease in bacteriophage, and both an integrase and an exonuclease from bacteria. The other four proteins are homologous to an endosymbiont-derived helicase, primase, single-stranded binding (SSB) protein, and thymidylate kinase, individually. Additionally, AbSV exhibits a common gene arrangement similar to the majority of bacteriophages. Unique to AbSV, the viral genome also contains genes associated with bacterial outer membrane proteins and may lack the structural protein-encoding ORFs. Genomic characterization of AbSV indicates that it may represent a transitional form of microbial evolution from viruses to bacteria. PMID:21079776

  11. Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha.

    PubMed

    Doki, Tomoyoshi; Takano, Tomomi; Hohdatsu, Tsutomu

    2016-10-01

    Feline infectious peritonitis (FIP) is a fatal inflammatory disease caused by FIP virus infection. Feline tumor necrosis factor (fTNF)-alpha is closely involved in the aggravation of FIP pathology. We previously described the preparation of neutralizing mouse anti-fTNF-alpha monoclonal antibody (mAb 2-4) and clarified its role in the clinical condition of cats with FIP using in vitro systems. However, administration of mouse mAb 2-4 to cat may lead to a production of feline anti-mouse antibodies. In the present study, we prepared a mouse-feline chimeric mAb (chimeric mAb 2-4) by fusing the variable region of mouse mAb 2-4 to the constant region of feline antibody. The chimeric mAb 2-4 was confirmed to have fTNF-alpha neutralization activity. Purified mouse mAb 2-4 and chimeric mAb 2-4 were repeatedly administered to cats, and the changes in the ability to induce feline anti-mouse antibody response were investigated. In the serum of cats treated with mouse mAb 2-4, feline anti-mouse antibody production was induced, and the fTNF-alpha neutralization effect of mouse mAb 2-4 was reduced. In contrast, in cats treated with chimeric mAb 2-4, the feline anti-mouse antibody response was decreased compared to that of mouse mAb 2-4-treated cats.

  12. Understanding Zika Virus Stability and Developing a Chimeric Vaccine through Functional Analysis

    PubMed Central

    Yang, Yujiao; Muruato, Antonio E.; Zou, Jing; Shan, Chao; Nunes, Bruno T. D.; Medeiros, Daniele B. A.; Vasconcelos, Pedro F. C.; Weaver, Scott C.; Rossi, Shannan L.

    2017-01-01

    ABSTRACT Compared with other flaviviruses, Zika virus (ZIKV) is uniquely associated with congenital diseases in pregnant women. One recent study reported that (i) ZIKV has higher thermostability than dengue virus (DENV [a flavivirus closely related to ZIKV]), which might contribute to the disease outcome; (ii) the higher thermostability of ZIKV could arise from an extended loop structure in domain III of the viral envelope (E) protein and an extra hydrogen-bond interaction between E molecules (V. A. Kostyuchenko, E. X. Y. Lim, S. Zhang, G. Fibriansah, T.-S. Ng, J. S. G. Ooi, J. Shi, and S.-M. Lok, Nature 533:425–428, 2016, https://doi.org/10.1038/nature17994). Here we report the functional analysis of the structural information in the context of complete ZIKV and DENV-2 virions. Swapping the prM-E genes between ZIKV and DENV-2 switched the thermostability of the chimeric viruses, identifying the prM-E proteins as the major determinants for virion thermostability. Shortening the extended loop of the E protein by 1 amino acid was lethal for ZIKV assembly/release. Mutations (Q350I and T351V) that abolished the extra hydrogen-bond interaction between the E proteins did not reduce ZIKV thermostability, indicating that the extra interaction does not increase the thermostability. Interestingly, mutant T351V was attenuated in A129 mice defective in type I interferon receptors, even though the virus retained the wild-type thermostability. Furthermore, we found that a chimeric ZIKV with the DENV-2 prM-E and a chimeric DENV-2 with the ZIKV prM-E were highly attenuated in A129 mice; these chimeric viruses were highly immunogenic and protective against DENV-2 and ZIKV challenge, respectively. These results indicate the potential of these chimeric viruses for vaccine development. PMID:28174309

  13. Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus

    PubMed Central

    2011-01-01

    Background To develop a plant-based vaccine against Plasmodium vivax, two P. vivax candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP), a component of sporozoites that contains a B-cell epitope. Methods A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR), beta-glucuronidase reporter gene (GUS) assay, and Western blot. Results The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n = 38) and a clinical specificity of 100% (n = 24) as assessed by enzyme-linked immunosorbent assay (ELISA). Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40), TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Conclusions The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria. PMID:21529346

  14. The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments

    SciTech Connect

    Zhang Fanglin; Wu Xingan; Luo Wen; Bai Wentao; Liu Yong; Yan Yan; Wang Haitao; Xu Zhikai . E-mail: zhikaixu@fmmu.edu.cn

    2007-03-23

    Hemorrhagic fever with renal syndrome (HFRS), which is characterized by severe symptoms and high mortality, is caused by hantavirus. There are still no effective prophylactic vaccines directed to HFRS until now. In this research, we fused expressed G2 fragment of M segment and 0.7 kb fragment of S segment. We expect it could be a candidate vaccine. Chimeric gene G2S0.7 was first expressed in prokaryotic expression system pGEX-4T. After inducing expressed fusion proteins, GST-G2S0.7 was induced and its molecular weight was about 100 kDa. Meanwhile, the fusion protein kept the activity of its parental proteins. Further, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response. The results showed that the chimeric gene could simultaneously evoke specific antibody against nucleocapsid protein (NP) and glycoprotein (GP). And the immunized mice of every group elicited neutralizing antibodies with different titers. But the titers were low. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to NP and GP were significantly higher than that of control. It suggested that the chimeric gene of Hantaan virus containing G2 fragment of M segment and 0.7 kb fragment of S segment could directly elicit specific anti-Hantaan virus humoral and cellular immune response in BALB/c mice.

  15. Chimeric Peptides as Implant Functionalization Agents for Titanium Alloy Implants with Antimicrobial Properties

    NASA Astrophysics Data System (ADS)

    Yucesoy, Deniz T.; Hnilova, Marketa; Boone, Kyle; Arnold, Paul M.; Snead, Malcolm L.; Tamerler, Candan

    2015-04-01

    Implant-associated infections can have severe effects on the longevity of implant devices and they also represent a major cause of implant failures. Treating these infections associated with implants by antibiotics is not always an effective strategy due to poor penetration rates of antibiotics into biofilms. Additionally, emerging antibiotic resistance poses serious concerns. There is an urge to develop effective antibacterial surfaces that prevent bacterial adhesion and proliferation. A novel class of bacterial therapeutic agents, known as antimicrobial peptides (AMPs), are receiving increasing attention as an unconventional option to treat septic infection, partly due to their capacity to stimulate innate immune responses and for the difficulty of microorganisms to develop resistance towards them. While host and bacterial cells compete in determining the ultimate fate of the implant, functionalization of implant surfaces with AMPs can shift the balance and prevent implant infections. In the present study, we developed a novel chimeric peptide to functionalize the implant material surface. The chimeric peptide simultaneously presents two functionalities, with one domain binding to a titanium alloy implant surface through a titanium-binding domain while the other domain displays an antimicrobial property. This approach gains strength through control over the bio-material interfaces, a property built upon molecular recognition and self-assembly through a titanium alloy binding domain in the chimeric peptide. The efficiency of chimeric peptide both in-solution and absorbed onto titanium alloy surface was evaluated in vitro against three common human host infectious bacteria, Streptococcus mutans, Staphylococcus epidermidis, and Escherichia coli. In biological interactions such as occur on implants, it is the surface and the interface that dictate the ultimate outcome. Controlling the implant surface by creating an interface composed chimeric peptides may therefore

  16. Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha

    PubMed Central

    DOKI, Tomoyoshi; TAKANO, Tomomi; HOHDATSU, Tsutomu

    2016-01-01

    Feline infectious peritonitis (FIP) is a fatal inflammatory disease caused by FIP virus infection. Feline tumor necrosis factor (fTNF)-alpha is closely involved in the aggravation of FIP pathology. We previously described the preparation of neutralizing mouse anti-fTNF-alpha monoclonal antibody (mAb 2–4) and clarified its role in the clinical condition of cats with FIP using in vitro systems. However, administration of mouse mAb 2–4 to cat may lead to a production of feline anti-mouse antibodies. In the present study, we prepared a mouse-feline chimeric mAb (chimeric mAb 2–4) by fusing the variable region of mouse mAb 2–4 to the constant region of feline antibody. The chimeric mAb 2–4 was confirmed to have fTNF-alpha neutralization activity. Purified mouse mAb 2–4 and chimeric mAb 2–4 were repeatedly administered to cats, and the changes in the ability to induce feline anti-mouse antibody response were investigated. In the serum of cats treated with mouse mAb 2–4, feline anti-mouse antibody production was induced, and the fTNF-alpha neutralization effect of mouse mAb 2–4 was reduced. In contrast, in cats treated with chimeric mAb 2–4, the feline anti-mouse antibody response was decreased compared to that of mouse mAb 2–4-treated cats. PMID:27264736

  17. Recombinant Mouse-Human Chimeric Antibodies as Calibrators in Immunoassays That Measure Antibodies to Toxoplasma gondii

    PubMed Central

    Hackett, John; Hoff-Velk, Jane; Golden, Alan; Brashear, Jeff; Robinson, John; Rapp, Margaret; Klass, Michael; Ostrow, David H.; Mandecki, Wlodek

    1998-01-01

    In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was ≥0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays. PMID:9574691

  18. Hybridization accompanying FRET event in labeled natural nucleoside-unnatural nucleoside containing chimeric DNA duplexes.

    PubMed

    Bag, Subhendu Sekhar; Das, Suman K; Pradhan, Manoj Kumar; Jana, Subhashis

    2016-09-01

    Förster resonance energy transfer (FRET) is a highly efficient strategy in illuminating the structures, structural changes and dynamics of DNA, proteins and other biomolecules and thus is being widely utilized in studying such phenomena, in designing molecular/biomolecular probes for monitoring the hybridization event of two single stranded DNA to form duplex, in gene detection and in many other sensory applications in chemistry, biology and material sciences. Moreover, FRET can give information about the positional status of chromophores within the associated biomolecules with much more accuracy than other methods can yield. Toward this end, we want to report here the ability of fluorescent unnatural nucleoside, triazolylphenanthrene ((TPhen)BDo) to show FRET interaction upon hybridization with fluorescently labeled natural nucleosides, (Per)U or (OxoPy)U or (Per)U, forming two stable chimeric DNA duplexes. The pairing selectivity and the thermal duplex stability of the chimeric duplexes are higher than any of the duplexes with natural nucleoside formed. The hybridization results in a Förster resonance energy transfer (FRET) from donor triazolylphenanthrene of (TPhen)BDo to acceptor oxopyrene of (OxoPy)U and/or to perylene chromophore of (Per)U, respectively, in two chimeric DNA duplexes. Therefore, we have established the FRET process in two chimeric DNA duplexes wherein a fluorescently labeled natural nucleoside ((OxoPy)U or (Per)U) paired against an unnatural nucleoside ((TPhen)BDo) without sacrificing the duplex stability and B-DNA conformation. The hybridization accompanying FRET event in these classes of interacting fluorophores is new. Moreover, there is no report of such designed system of chimeric DNA duplex. Our observed phenomenon and the design can potentially be exploited in designing more of such efficient FRET pairs for useful application in the detection and analysis of biomolecular interactions and in material science application. Copyright

  19. Temporally chimeric mice reveal flexibility of circadian period-setting in the suprachiasmatic nucleus

    PubMed Central

    Smyllie, Nicola J.; Chesham, Johanna E.; Hamnett, Ryan; Maywood, Elizabeth S.; Hastings, Michael H.

    2016-01-01

    The suprachiasmatic nucleus (SCN) is the master circadian clock controlling daily behavior in mammals. It consists of a heterogeneous network of neurons, in which cell-autonomous molecular feedback loops determine the period and amplitude of circadian oscillations of individual cells. In contrast, circuit-level properties of coherence, synchrony, and ensemble period are determined by intercellular signals and are embodied in a circadian wave of gene expression that progresses daily across the SCN. How cell-autonomous and circuit-level mechanisms interact in timekeeping is poorly understood. To explore this interaction, we used intersectional genetics to create temporally chimeric mice with SCN containing dopamine 1a receptor (Drd1a) cells with an intrinsic period of 24 h alongside non-Drd1a cells with 20-h clocks. Recording of circadian behavior in vivo alongside cellular molecular pacemaking in SCN slices in vitro demonstrated that such chimeric circuits form robust and resilient circadian clocks. It also showed that the computation of ensemble period is nonlinear. Moreover, the chimeric circuit sustained a wave of gene expression comparable to that of nonchimeric SCN, demonstrating that this circuit-level property is independent of differences in cell-intrinsic periods. The relative dominance of 24-h Drd1a and 20-h non-Drd1a neurons in setting ensemble period could be switched by exposure to resonant or nonresonant 24-h or 20-h lighting cycles. The chimeric circuit therefore reveals unanticipated principles of circuit-level operation underlying the emergent plasticity, resilience, and robustness of the SCN clock. The spontaneous and light-driven flexibility of period observed in chimeric mice provides a new perspective on the concept of SCN pacemaker cells. PMID:26966234

  20. Expression of a chimeric human/salmon calcitonin gene integrated into the Saccharomyces cerevisiae genome using rDNA sequences as recombination sites.

    PubMed

    Sun, Hengyi; Zang, Xiaonan; Liu, Yuantao; Cao, Xiaofei; Wu, Fei; Huang, Xiaoyun; Jiang, Minjie; Zhang, Xuecheng

    2015-12-01

    Calcitonin participates in controlling homeostasis of calcium and phosphorus and plays an important role in bone metabolism. The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to express chimeric human/salmon calcitonin (hsCT) without the use of antibiotics. To do so, a homologous recombination plasmid pUC18-rDNA2-ura3-P pgk -5hsCT-rDNA1 was constructed, which contains two segments of ribosomal DNA of 1.1 kb (rDNA1) and 1.4 kb (rDNA2), to integrate the heterologous gene into host rDNA. A DNA fragment containing five copies of a chimeric human/salmon calcitonin gene (5hsCT) under the control of the promoter for phosphoglycerate kinase (P pgk ) was constructed to express 5hsCT in S. cerevisiae using ura3 as a selectable auxotrophic marker gene. After digestion by restriction endonuclease HpaI, a linear fragment, rDNA2-ura3-P pgk -5hsCT-rDNA1, was obtained and transformed into the △ura3 mutant of S. cerevisiae by the lithium acetate method. The ura3-P pgk -5hsCT sequence was introduced into the genome at rDNA sites by homologous recombination, and the recombinant strain YS-5hsCT was obtained. Southern blot analysis revealed that the 5hsCT had been integrated successfully into the genome of S. cerevisiae. The results of Western blot and ELISA confirmed that the 5hsCT protein had been expressed in the recombinant strain YS-5hsCT. The expression level reached 2.04 % of total proteins. S. cerevisiae YS-5hsCT decreased serum calcium in mice by oral administration and even 0.01 g lyophilized S. cerevisiae YS-5hsCT/kg decreased serum calcium by 0.498 mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis.

  1. Impact of Mixed Xenogeneic Porcine Hematopoietic Chimerism on Human NK Cell Recognition in a Humanized Mouse Model.

    PubMed

    Li, H W; Vishwasrao, P; Hölzl, M A; Chen, S; Choi, G; Zhao, G; Sykes, M

    2017-02-01

    Mixed chimerism is a promising approach to inducing allograft and xenograft tolerance. Mixed allogeneic and xenogeneic chimerism in mouse models induced specific tolerance and global hyporesponsiveness, respectively, of host mouse natural killer (NK) cells. In this study, we investigated whether pig/human mixed chimerism could tolerize human NK cells in a humanized mouse model. Our results showed no impact of induced human NK cell reconstitution on porcine chimerism. NK cells from most pig/human mixed chimeric mice showed either specifically decreased cytotoxicity to pig cells or global hyporesponsiveness in an in vitro cytotoxicity assay. Mixed xenogeneic chimerism did not hamper the maturation of human NK cells but was associated with an alteration in NK cell subset distribution and interferon gamma (IFN-γ) production in the bone marrow. In summary, we demonstrate that mixed xenogeneic chimerism induces human NK cell hyporesponsiveness to pig cells. Our results support the use of this approach to inducing xenogeneic tolerance in the clinical setting. However, additional approaches are required to improve the efficacy of tolerance induction while ensuring adequate NK cell functions.

  2. Single copy DNA homology in sea stars.

    PubMed

    Smith, M J; Nicholson, R; Stuerzl, M; Lui, A

    1982-01-01

    The sequence homology in the single copy DNA of sea stars has been measured. Labeled single copy DNA from Pisaster ochraceus was reannealed with excess genomic DNA from P. brevispinus, Evasterias troschelii, Pycnopodia helianthoides, Solaster stimpsoni, and Dermasterias imbricata. Reassociation reactions were performed under two criteria of salt and temperature. The extent of reassociation and thermal denaturation characteristics of hybrid single copy DNA molecules follow classical taxonomic lines. P. brevispinus DNA contains essentially all of the sequences present in P. ochraceus single copy tracer while Evasterias and Pycnopodia DNAs contain 52% and 46% of such sequences respectively. Reciprocal reassociation reactions with labeled Evasterias single copy DNA confirm the amount and fidelity of the sequence homology. There is a small definite reaction of uncertain homology between P. ochraceus single copy DNA and Solaster or Dermasterias DNA. Similarly Solaster DNA contains sequences homologous to approximately 18% of Dermasterias unique DNA. The thermal denaturation temperatures of heteroduplexes indicate that the genera Pisaster and Evasterias diverged shortly after the divergence of the subfamilies Pycnopodiinae and Asteriinae. The two Pisaster species diverged more recently, probably in the most recent quarter of the interval since the separation of the genera Pisaster and Evasterias.

  3. Cyclic homology for Hom-associative algebras

    NASA Astrophysics Data System (ADS)

    Hassanzadeh, Mohammad; Shapiro, Ilya; Sütlü, Serkan

    2015-12-01

    In the present paper we investigate the noncommutative geometry of a class of algebras, called the Hom-associative algebras, whose associativity is twisted by a homomorphism. We define the Hochschild, cyclic, and periodic cyclic homology and cohomology for this class of algebras generalizing these theories from the associative to the Hom-associative setting.

  4. Biochemistry of homologous recombination in Escherichia coli.

    PubMed Central

    Kowalczykowski, S C; Dixon, D A; Eggleston, A K; Lauder, S D; Rehrauer, W M

    1994-01-01

    Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination. Images PMID:7968921

  5. Persistent homology in graph power filtrations

    PubMed Central

    Marchette, David J.

    2016-01-01

    The persistence of homological features in simplicial complex representations of big datasets in Rn resulting from Vietoris–Rips or Čech filtrations is commonly used to probe the topological structure of such datasets. In this paper, the notion of homological persistence in simplicial complexes obtained from power filtrations of graphs is introduced. Specifically, the rth complex, r ≥ 1, in such a power filtration is the clique complex of the rth power Gr of a simple graph G. Because the graph distance in G is the relevant proximity parameter, unlike a Euclidean filtration of a dataset where regional scale differences can be an issue, persistence in power filtrations provides a scale-free insight into the topology of G. It is shown that for a power filtration of G, the girth of G defines an r range over which the homology of the complexes in the filtration are guaranteed to persist in all dimensions. The role of chordal graphs as trivial homology delimiters in power filtrations is also discussed and the related notions of ‘persistent triviality’, ‘transient noise’ and ‘persistent periodicity’ in power filtrations are introduced. PMID:27853540

  6. Rad54, the Motor of Homologous Recombination

    PubMed Central

    Mazin, Alexander V.; Mazina, Olga M.; Bugreev, Dmitry V.; Rossi, Matthew J.

    2009-01-01

    Homologous recombination (HR) performs crucial functions including DNA repair, segregation of homologous chromosomes, propagation of genetic diversity, and maintenance of telomeres. HR is responsible for the repair of DNA double-strand breaks and DNA interstrand cross-links. The process of HR is initiated at the site of DNA breaks and gaps and involves a search for homologous sequences promoted by Rad51 and auxiliary proteins followed by the subsequent invasion of broken DNA ends into the homologous duplex DNA that then serves as a template for repair. The invasion produces a cross-stranded structure, known as the Holliday junction. Here, we describe the properties of Rad54, an important and versatile HR protein that is evolutionarily conserved in eukaryotes. Rad54 is a motor protein that translocates along dsDNA and performs several important functions in HR. The current review focuses on the recently identified Rad54 activities which contribute to the late phase of HR, especially the branch migration of Holliday junctions. PMID:20089461

  7. Homology modeling of human muscarinic acetylcholine receptors.

    PubMed

    Thomas, Trayder; McLean, Kimberley C; McRobb, Fiona M; Manallack, David T; Chalmers, David K; Yuriev, Elizabeth

    2014-01-27

    We have developed homology models of the acetylcholine muscarinic receptors M₁R-M₅R, based on the β₂-adrenergic receptor crystal as the template. This is the first report of homology modeling of all five subtypes of acetylcholine muscarinic receptors with binding sites optimized for ligand binding. The models were evaluated for their ability to discriminate between muscarinic antagonists and decoy compounds using virtual screening using enrichment factors, area under the ROC curve (AUC), and an early enrichment measure, LogAUC. The models produce rational binding modes of docked ligands as well as good enrichment capacity when tested against property-matched decoy libraries, which demonstrates their unbiased predictive ability. To test the relative effects of homology model template selection and the binding site optimization procedure, we generated and evaluated a naïve M₂R model, using the M₃R crystal structure as a template. Our results confirm previous findings that binding site optimization using ligand(s) active at a particular receptor, i.e. including functional knowledge into the model building process, has a more pronounced effect on model quality than target-template sequence similarity. The optimized M₁R-M₅R homology models are made available as part of the Supporting Information to allow researchers to use these structures, compare them to their own results, and thus advance the development of better modeling approaches.

  8. Construction and characterization of a chimeric virus (BIV/HIV-1) carrying the bovine immunodeficiency virus gag-pol gene.

    PubMed

    Chen, Guomin; Wang, Shuhui; Xiong, Kun; Wang, Jinzhong; Ye, Tao; Dong, Wenping; Wang, Qi; Chen, Qimin; Geng, Yunqi; Wood, Charles; Zeng, Yi

    2002-01-04

    HIV-1(HXB2) 5'LTR region, most of BIV(R29) gag-pol segment and HIV-1(HXB2) pol IN-3'LTR region were respectively amplified. A chimeric clone, designated as pHBIV(3753), was constructed by cloning three fragments sequentially into pUC18. MT4 cells were transfected with pHBIV3753. The replication and expressions of the chimeric virus (HBIV3753) were monitored by RT activity and IFA. The results firstly demonstrated that it is possible to generate a new type of the BIV/HIV-1 chimeric virus containing BIV gag-pol gene.

  9. Understanding Zika Virus Stability and Developing a Chimeric Vaccine through Functional Analysis.

    PubMed

    Xie, Xuping; Yang, Yujiao; Muruato, Antonio E; Zou, Jing; Shan, Chao; Nunes, Bruno T D; Medeiros, Daniele B A; Vasconcelos, Pedro F C; Weaver, Scott C; Rossi, Shannan L; Shi, Pei-Yong

    2017-02-07

    Compared with other flaviviruses, Zika virus (ZIKV) is uniquely associated with congenital diseases in pregnant women. One recent study reported that (i) ZIKV has higher thermostability than dengue virus (DENV [a flavivirus closely related to ZIKV]), which might contribute to the disease outcome; (ii) the higher thermostability of ZIKV could arise from an extended loop structure in domain III of the viral envelope (E) protein and an extra hydrogen-bond interaction between E molecules (V. A. Kostyuchenko, E. X. Y. Lim, S. Zhang, G. Fibriansah, T.-S. Ng, J. S. G. Ooi, J. Shi, and S.-M. Lok, Nature 533:425-428, 2016, https://doi.org/10.1038/nature17994). Here we report the functional analysis of the structural information in the context of complete ZIKV and DENV-2 virions. Swapping the prM-E genes between ZIKV and DENV-2 switched the thermostability of the chimeric viruses, identifying the prM-E proteins as the major determinants for virion thermostability. Shortening the extended loop of the E protein by 1 amino acid was lethal for ZIKV assembly/release. Mutations (Q350I and T351V) that abolished the extra hydrogen-bond interaction between the E proteins did not reduce ZIKV thermostability, indicating that the extra interaction does not increase the thermostability. Interestingly, mutant T351V was attenuated in A129 mice defective in type I interferon receptors, even though the virus retained the wild-type thermostability. Furthermore, we found that a chimeric ZIKV with the DENV-2 prM-E and a chimeric DENV-2 with the ZIKV prM-E were highly attenuated in A129 mice; these chimeric viruses were highly immunogenic and protective against DENV-2 and ZIKV challenge, respectively. These results indicate the potential of these chimeric viruses for vaccine development. Analysis of a recently observed high-resolution structure of ZIKV led to a hypothesis that its unusual stability may contribute to the associated, unique disease outcomes. Here we performed a functional

  10. Increased chromosome mobility facilitates homology search during recombination.

    PubMed

    Miné-Hattab, Judith; Rothstein, Rodney

    2012-04-08

    Homologous recombination, an essential process for preserving genomic integrity, uses intact homologous sequences to repair broken chromosomes. To explore the mechanism of homologous pairing in vivo, we tagged two homologous loci in diploid yeast Saccharomyces cerevisiae cells and investigated their dynamic organization in the absence and presence of DNA damage. When neither locus is damaged, homologous loci occupy largely separate regions, exploring only 2.7% of the nuclear volume. Following the induction of a double-strand break, homologous loci co-localize ten times more often. The mobility of the cut chromosome markedly increases, allowing it to explore a nuclear volume that is more than ten times larger. Interestingly, the mobility of uncut chromosomes also increases, allowing them to explore a four times larger volume. We propose a model for homology search in which increased chromosome mobility facilitates homologous pairing. Finally, we find that the increase in DNA dynamics is dependent on early steps of homologous recombination.

  11. Delayed ripening and improved fruit processing quality in tomato by RNAi-mediated silencing of three homologs of 1-aminopropane-1-carboxylate synthase gene.

    PubMed

    Gupta, Aarti; Pal, Ram Krishna; Rajam, Manchikatla Venkat

    2013-07-15

    The ripening hormone, ethylene is known to initiate, modulate and co-ordinate the expression of various genes involved in the ripening process. The burst in ethylene production is the key event for the onset of ripening in climacteric fruits, including tomatoes. Therefore ethylene is held accountable for the tons of post-harvest losses due to over-ripening and subsequently resulting in fruit rotting. In the present investigation, delayed ripening tomatoes were generated by silencing three homologs of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) gene during the course of ripening using RNAi technology. The chimeric RNAi-ACS construct designed to target ACS homologs, effectively repressed the ethylene production in tomato fruits. Fruits from such lines exhibited delayed ripening and extended shelf life for ∼45 days, with improved juice quality. The ethylene suppression brought about compositional changes in these fruits by enhancing polyamine (PA) levels. Further, decreased levels of ethylene in RNAi-ACS fruits has led to the altered levels of various ripening-specific transcripts, especially the up-regulation of PA biosynthesis and ascorbic acid (AsA) metabolism genes and down-regulation of cell wall hydrolyzing enzyme genes. These results suggest that the down-regulation of ACS homologs using RNAi can be an effective approach for obtaining delayed ripening with longer shelf life and an enhanced processing quality of tomato fruits. Also, the chimeric gene fusion can be used as an effective design for simultaneous silencing of more than one gene. These observations would be useful in better understanding of the ethylene and PA signaling during fruit ripening and molecular mechanisms underlying the interaction of these two molecules in affecting fruit quality traits. Copyright © 2013 Elsevier GmbH. All rights reserved.

  12. Early mixed T-cell chimerism is predictive of pediatric AML or MDS relapse after hematopoietic stem cell transplant.

    PubMed

    Broglie, Larisa; Helenowski, Irene; Jennings, Lawrence J; Schafernak, Kristian; Duerst, Reggie; Schneiderman, Jennifer; Tse, William; Kletzel, Morris; Chaudhury, Sonali

    2017-03-07

    Patients with acute myeloid leukemia (AML) who relapse after hematopoietic stem cell transplantation (HCT) have dismal outcomes. Our ability to predict those at risk for relapse is limited. We examined chimerism trends post-HCT in 63 children who underwent HCT for AML or myelodysplastic syndrome (MDS). Mixed T-cell chimerism at engraftment and absence of chronic graft versus host disease (cGVHD) were associated with relapse (P = 0.04 and P = 0.02, respectively). Mixed T-cell chimerism at engraftment was predictive in patients without cGVHD (P = 0.03). Patients with engraftment mixed T-cell chimerism may warrant closer disease monitoring and consideration for early intervention.

  13. Flow cytometric evaluation of red blood cell chimerism after bone marrow transplantation in Iranian patients: a preliminary study.

    PubMed

    Shaiegan, Mojgan; Hadjati, Esmerdis; Aghaiipour, Mahnaz; Iravani, Masoud; David, Gaelle; Bernard, Daniel

    2006-10-01

    The aim of this study was to evaluate mixed red cells population and red blood cell chimerism after hematopoietic stem cell transplantation. Red blood cell chimerism after hematopoietic stem cell transplantation was analyzed using a series of fluorescein isothiocyanate-conjugated monoclonal antibodies (BioAtlantic, France) directed against ABH, Rh (D, C, E, c, e), Kell, Duffy, Kidd, and Ss antigens on blood samples of 14 patients with hematologic disorders undergoing hematopoietic stem cell transplantation, by flow cytometric method on days 15, 30, and 60 after transplantation. All patients showed expression of donor red cell antigens within days 15 - 30 after hematopoietic stem cell transplantation. Graft versus host disease and ABO incompatibility did not affect the expression of chimerism. Flow cytometric analysis is a simple, accurate, and valuable test which is of significant help in monitoring chimerism in allogeneic hematopoietic stem cell transplantation.

  14. Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication

    PubMed Central

    Gagnon, David; Lehoux, Michaël

    2015-01-01

    ABSTRACT The E1 helicase from anogenital human papillomavirus (HPV) types interacts with the cellular WD repeat-containing protein UAF1 in complex with the deubiquitinating enzyme USP1, USP12, or USP46. This interaction stimulates viral DNA replication and is required for maintenance of the viral episome in keratinocytes. E1 associates with UAF1 through a short UAF1-binding site (UBS) located within the N-terminal 40 residues of the protein. Here, we investigated if the E1 UBS could be replaced by the analogous domain from an unrelated protein, the pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). We found that PHLPP1 and E1 interact with UAF1 in a mutually exclusive manner and mapped the minimal PHLPP1 UBS (PUBS) to a 100-amino-acid region sufficient for assembly into UAF1-USP complexes. Similarly to the E1 UBS, overexpression of PUBS in trans inhibited HPV DNA replication, albeit less efficiently. Characterization of a PHLPP1-E1 chimeric helicase revealed that PUBS could partially substitute for the E1 UBS in enhancing viral DNA replication and that the stimulatory effect of PUBS likely involves recruitment of UAF1-USP complexes, as it was abolished by mutations that weaken UAF1-binding and by overexpression of catalytically inactive USPs. Although functionally similar to the E1 UBS, PUBS is larger in size and requires both the WD repeat region and C-terminal ubiquitin-like domain of UAF1 for interaction, in contrast to E1, which does not contact the latter. Overall, this comparison of two heterologous UBSs indicates that these domains function as transferable protein interaction modules and provide further evidence that the association of E1 with UAF1-containing deubiquitinating complexes stimulates HPV DNA replication. IMPORTANCE The E1 protein from anogenital HPV types interacts with the UAF1-associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome. Little is known about the

  15. Text mining of DNA sequence homology searches.

    PubMed

    McCallum, John; Ganesh, Siva

    2003-01-01

    Primary tasks in analysis and annotation of expressed sequence tag (EST) datasets are to identify similarity among sequences by unsupervised clustering and assign putative function based on BLAST homology searches. We investigated the usefulness of text mining as a simple approach for further higher-level clustering of EST datasets using IBM Intelligent Miner for Text v2.3 tools. Agglomerative and k-means clustering tools were used to cluster BLASTx homology search documents from two onion EST datasets and optimised by pre-processing and pruning. Subjective evaluation confirmed that these tools provided biologically useful and complementary views of the two libraries, provided new insights into their composition and revealed clusters previously identified by human experts. We compared BLASTx textual clusters for two gene families with their DNA sequence-based clusters and confirmed that these shared similar morphology.

  16. Redesigning Aldolase Stereoselectivity by Homologous Grafting

    PubMed Central

    Henßen, Birgit; Metz, Alexander; Gohlke, Holger; Pietruszka, Jörg

    2016-01-01

    The 2-deoxy-d-ribose-5-phosphate aldolase (DERA) offers access to highly desirable building blocks for organic synthesis by catalyzing a stereoselective C-C bond formation between acetaldehyde and certain electrophilic aldehydes. DERA´s potential is particularly highlighted by the ability to catalyze sequential, highly enantioselective aldol reactions. However, its synthetic use is limited by the absence of an enantiocomplementary enzyme. Here, we introduce the concept of homologous grafting to identify stereoselectivity-determining amino acid positions in DERA. We identified such positions by structural analysis of the homologous aldolases 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG) and the enantiocomplementary enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase (KDPGal). Mutation of these positions led to a slightly inversed enantiopreference of both aldolases to the same extent. By transferring these sequence motifs onto DERA we achieved the intended change in enantioselectivity. PMID:27327271

  17. Homologation Reaction of Ketones with Diazo Compounds.

    PubMed

    Candeias, Nuno R; Paterna, Roberta; Gois, Pedro M P

    2016-03-09

    This review covers the addition of diazo compounds to ketones to afford homologated ketones, either in the presence or in the absence of promoters or catalysts. Reactions with diazoalkanes, aryldiazomethanes, trimethylsilyldiazomethane, α-diazo esters, and disubstituted diazo compounds are covered, commenting on the complex regiochemistry of the reaction and the nature of the catalysts and promoters. The recent reports on the enantioselective version of ketone homologation reactions are gathered in one section, followed by reports on the use of cyclic ketones ring expansion in total synthesis. Although the first reports of this reaction appeared in the literature almost one century ago, the recent achievements, in particular, for the asymmetric version, forecast the development of new breakthroughs in the synthetically valuable field of diazo chemistry.

  18. Homologous Pairing between Long DNA Double Helices

    NASA Astrophysics Data System (ADS)

    Mazur, Alexey K.

    2016-04-01

    Molecular recognition between two double stranded (ds) DNA with homologous sequences may not seem compatible with the B-DNA structure because the sequence information is hidden when it is used for joining the two strands. Nevertheless, it has to be invoked to account for various biological data. Using quantum chemistry, molecular mechanics, and hints from recent genetics experiments, I show here that direct recognition between homologous dsDNA is possible through the formation of short quadruplexes due to direct complementary hydrogen bonding of major-groove surfaces in parallel alignment. The constraints imposed by the predicted structures of the recognition units determine the mechanism of complexation between long dsDNA. This mechanism and concomitant predictions agree with the available experimental data and shed light upon the sequence effects and the possible involvement of topoisomerase II in the recognition.

  19. Homologous pairing in stretched supercoiled DNA

    PubMed Central

    Strick, T. R.; Croquette, V.; Bensimon, D.

    1998-01-01

    By using elastic measurements on single DNA molecules, we show that stretching a negatively supercoiled DNA activates homologous pairing in physiological conditions. These experiments indicate that a stretched unwound DNA locally denatures to alleviate the force-driven increase in torsional stress. This is detected by hybridization with 1 kb of homologous single-stranded DNA probes. The stretching force involved (≈2 pN) is small compared with those typically developed by molecular motors, suggesting that this process may be relevant to DNA processing in vivo. We used this technique to monitor the progressive denaturation of DNA as it is unwound and found that distinct, stable denaturation bubbles formed, beginning in A+T-rich regions. PMID:9724746

  20. Effects of mixed hematopoietic chimerism in a mouse model of bone marrow transplantation for sickle cell anemia.

    PubMed

    Iannone, R; Luznik, L; Engstrom, L W; Tennessee, S L; Askin, F B; Casella, J F; Kickler, T S; Goodman, S N; Hawkins, A L; Griffin, C A; Noffsinger, L; Fuchs, E J

    2001-06-15

    Sickle cell anemia (SCA) is an inherited disorder of beta-globin, resulting in red blood cell rigidity, anemia, painful crises, organ infarctions, and reduced life expectancy. Allogeneic blood or marrow transplantation (BMT) can cure SCA but is associated with an 8% to 10% mortality rate, primarily from complications of marrow-ablative conditioning. Transplantation of allogeneic marrow after less intensive conditioning reduces toxicity but may result in stable mixed hematopoietic chimerism. The few SCA patients who inadvertently developed mixed chimerism after BMT remain symptom free, suggesting that mixed chimerism can reduce disease-related morbidity. However, because the effects of various levels of mixed chimerism on organ pathology have not been characterized, this study examined the histologic effects of an increasing percentage of normal donor hematopoiesis in a mouse model of BMT for SCA. In lethally irradiated normal mice that were reconstituted with varying ratios of T-cell-depleted marrow from normal and transgenic "sickle cell" mice, normal myeloid chimerism in excess of 25% was associated with more than 90% normal hemoglobin (Hb). However, 70% normal myeloid chimerism was required to reverse the anemia. Organ pathology, including liver infarction, was present in mice with sickle Hb (HbS) levels as low as 16.8% (19.6% normal myeloid chimerism). Histologic abnormalities increased in severity up to 80% HbS, but were less severe in mice with more than 80% HbS than in those with 40% to 80% HbS. Therefore, stable mixed chimerism resulting from nonmyeloablative BMT may reduce the morbidity from SCA, but prevention of all disease complications may require minimizing the fraction of circulating sickle red cells. (Blood. 2001;97:3960-3965)

  1. Homology and phylogeny and their automated inference

    NASA Astrophysics Data System (ADS)

    Fuellen, Georg

    2008-06-01

    The analysis of the ever-increasing amount of biological and biomedical data can be pushed forward by comparing the data within and among species. For example, an integrative analysis of data from the genome sequencing projects for various species traces the evolution of the genomes and identifies conserved and innovative parts. Here, I review the foundations and advantages of this “historical” approach and evaluate recent attempts at automating such analyses. Biological data is comparable if a common origin exists (homology), as is the case for members of a gene family originating via duplication of an ancestral gene. If the family has relatives in other species, we can assume that the ancestral gene was present in the ancestral species from which all the other species evolved. In particular, describing the relationships among the duplicated biological sequences found in the various species is often possible by a phylogeny, which is more informative than homology statements. Detecting and elaborating on common origins may answer how certain biological sequences developed, and predict what sequences are in a particular species and what their function is. Such knowledge transfer from sequences in one species to the homologous sequences of the other is based on the principle of ‘my closest relative looks and behaves like I do’, often referred to as ‘guilt by association’. To enable knowledge transfer on a large scale, several automated ‘phylogenomics pipelines’ have been developed in recent years, and seven of these will be described and compared. Overall, the examples in this review demonstrate that homology and phylogeny analyses, done on a large (and automated) scale, can give insights into function in biology and biomedicine.

  2. Khovanov homology of graph-links

    SciTech Connect

    Nikonov, Igor M

    2012-08-31

    Graph-links arise as the intersection graphs of turning chord diagrams of links. Speaking informally, graph-links provide a combinatorial description of links up to mutations. Many link invariants can be reformulated in the language of graph-links. Khovanov homology, a well-known and useful knot invariant, is defined for graph-links in this paper (in the case of the ground field of characteristic two). Bibliography: 14 titles.

  3. Dual submanifolds in rational homology spheres

    NASA Astrophysics Data System (ADS)

    Fang, FuQuan

    2017-09-01

    Let $\\Sigma$ be a simply connected rational homology sphere. A pair of disjoint closed submanifolds $M_+, M_-$ in $\\Sigma$ are called dual to each other if the complement $\\Sigma - M_+$ strongly homotopy retracts onto $M_-$ or vice-versa. In this paper we will give a complete answer of which integral triples $(n; m_+, m_-)$ can appear, where $n=dim \\Sigma -1$, $m_+={codim}M_+ -1$ and $m_-={codim}M_- -1$.

  4. Functional expression and properties of a nicotinic alpha9/5-HT3A chimeric receptor.

    PubMed

    Verbitsky, Miguel; Plazas, Paola V; Elgoyhen, A Belén

    2003-10-27

    We describe the functional properties of a nicotinic alpha9/serotonin subtype 3A (5HT3A) chimeric receptor expressed in Xenopus laevis oocytes. The chimera preserved ligand-binding properties of alpha9 and channel properties of 5HT3A. Thus, it responded to acetylcholine in a concentration-dependent manner with an EC50 of 70 microM but not to serotonin. It was blocked by methyllycaconitine, strychnine, atropine and nicotine, with the same rank order of potency as alpha9 receptors. The current-voltage relationship of currents through the alpha9/5HT3A chimera was similar to that of the 5HT3A receptors. These results are an evidence of functional coupling between the ligand-binding and the channel domains of the chimeric receptor.

  5. Expression of a chimeric CSF1R-LTK mediates ligand-dependent neurite outgrowth.

    PubMed

    Yamada, Shigeru; Nomura, Takashi; Takano, Kota; Fujita, Satoshi; Miyake, Masato; Miyake, Jun

    2008-11-19

    In an earlier screening, we identified several genes for kinases that might control the extension of neurites. One of these genes encoded a leukocyte tyrosine kinase (LTK), which is a receptor tyrosine kinase whose ligands remain to be identified. To examine the possible role of this LTK in neurite outgrowth, we constructed a chimeric receptor, in which the extracellular domain of the receptor for colony-stimulating factor-1 was fused to the cytoplasmic domain of LTK, which allowed the selective activation of LTK by colony-stimulating factor-1. Our studies using this chimeric receptor suggest that activation of the tyrosine kinase activity of LTK is sufficient to promote neurite outgrowth through pathways that include reactions catalyzed by phosphatidylinositol 3-kinase and MAPK.

  6. Association of pigmentary anomalies with chromosomal and genetic mosaicism and chimerism.

    PubMed Central

    Thomas, I T; Frias, J L; Cantu, E S; Lafer, C Z; Flannery, D B; Graham, J G

    1989-01-01

    We have evaluated eight patients with pigmentary anomalies reminiscent of incontinentia pigmenti or hypomelanosis of Ito. All demonstrated abnormal lymphocyte karyotypes with chromosomal mosaicism in lymphocytes and/or skin fibroblasts. In seven the skin was darkly pigmented, and in all of these seven cases the abnormal pigmentation followed Blaschko lines. The literature contains at least 36 similar examples of an association between pigmentary anomalies and chromosomal mosaicism, as well as five examples of an association with chimerism. The pigmentary anomalies are pleomorphic, and the chromosomal anomalies involve autosomes and sex chromosomes. The pigmentation patterns are reminiscent of the archetypal paradigm seen in allophenic mice and demonstrate the clonal origin of melanoblasts from neural crest precursors. Patients with anomalous skin pigmentation, particularly when it follows a pattern of Blaschko lines, should be appropriately evaluated for a possible association with chromosomal or genetic mosaicism or chimerism. Images Figure 1 PMID:2667350

  7. [Chimerism analysis after allogeneic haematopoietic stem cell transplantation. Interest of cell sorting: general review].

    PubMed

    Mollet, I; Giannoli, C; Rigal, D; Dubois, V

    2012-04-01

    Haematopoietic stem cells transplantation, widely used these last decades, represent the ultimate treatment resource for patients with haematological malignancies. Long range success of this treatment is particularly affected by relapse of the initial disease, graft rejection or graft versus host disease. Chimerism analysis after transplantation had been used since several years to document engraftment, to determine the risk of relapse and to adapt therapy promptly when necessary. Usefulness of this analysis for the outcome of transplanted patients, as well as the impact of using high sensitive techniques coupled with specific cell populations sorted have been demonstrated by retrospective studies. Follow-up of chimerism would allow to operate efficiently before the onset of clinical signs in leukaemic patients with high risk of relapse and to control the expression of minimal residual disease when specific molecular markers could not be monitored. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

  8. Birth of Four Chimeric Plastid Gene Clusters in Japanese Umbrella Pine

    PubMed Central

    Hsu, Chih-Yao; Wu, Chung-Shien; Chaw, Shu-Miaw

    2016-01-01

    Many genes in the plastid genomes (plastomes) of plants are organized as gene clusters, in which genes are co-transcribed, resembling bacterial operons. These plastid operons are highly conserved, even among conifers, whose plastomes are highly rearranged relative to other seed plants. We have determined the complete plastome sequence of Sciadopitys verticillata (Japanese umbrella pine), the sole member of Sciadopityaceae. The Sciadopitys plastome is characterized by extensive inversions, pseudogenization of four tRNA genes after tandem duplications, and a unique pair of 370-bp inverted repeats involved in the formation of isomeric plastomes. We showed that plastomic inversions in Sciadopitys have led to shuffling of the remote conserved operons, resulting in the birth of four chimeric gene clusters. Our data also demonstrated that the relocated genes can be co-transcribed in these chimeric gene clusters. The plastome of Sciadopitys advances our current understanding of how the conifer plastomes have evolved toward increased diversity and complexity. PMID:27269365

  9. Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease.

    PubMed

    Ellebrecht, Christoph T; Bhoj, Vijay G; Nace, Arben; Choi, Eun Jung; Mao, Xuming; Cho, Michael Jeffrey; Di Zenzo, Giovanni; Lanzavecchia, Antonio; Seykora, John T; Cotsarelis, George; Milone, Michael C; Payne, Aimee S

    2016-07-08

    Ideally, therapy for autoimmune diseases should eliminate pathogenic autoimmune cells while sparing protective immunity, but feasible strategies for such an approach have been elusive. Here, we show that in the antibody-mediated autoimmune disease pemphigus vulgaris (PV), autoantigen-based chimeric immunoreceptors can direct T cells to kill autoreactive B lymphocytes through the specificity of the B cell receptor (BCR). We engineered human T cells to express a chimeric autoantibody receptor (CAAR), consisting of the PV autoantigen, desmoglein (Dsg) 3, fused to CD137-CD3ζ signaling domains. Dsg3 CAAR-T cells exhibit specific cytotoxicity against cells expressing anti-Dsg3 BCRs in vitro and expand, persist, and specifically eliminate Dsg3-specific B cells in vivo. CAAR-T cells may provide an effective and universal strategy for specific targeting of autoreactive B cells in antibody-mediated autoimmune disease.

  10. A Photinus pyralis and Luciola italica chimeric firefly luciferase produces enhanced bioluminescence.

    PubMed

    Branchini, Bruce R; Southworth, Tara L; Fontaine, Danielle M; Davis, Audrey L; Behney, Curran E; Murtiashaw, Martha H

    2014-10-14

    We report the enhanced bioluminescence properties of a chimeric enzyme (PpyLit) that contains the N-domain of recombinant Photinus pyralis luciferase joined to the C-domain of recombinant Luciola italica luciferase. Compared to the P. pyralis enzyme, the novel PpyLit chimera exhibited 1.8-fold enhanced flash-height specific activity, 2.0-fold enhanced integration-based specific activity, 2.9-fold enhanced catalytic efficiency (kcat/Km), and a 1.4-fold greater bioluminescence quantum yield. The results of this study provide an underlying basis of this unusual example of a chimeric enzyme with enhanced catalytic properties that are not simply the sum of the contributions of the two luciferases.

  11. Chimeric Antigen Receptor-Redirected T cells return to the bench

    PubMed Central

    Geldres, Claudia; Savoldo, Barbara; Dotti, Gianpietro

    2016-01-01

    While the clinical progress of chimeric antigen receptor T cell (CAR-T) immunotherapy has garnered attention to the field, our understanding of the biology of these chimeric molecules is still emerging. Our aim within this review is to bring to light the mechanistic understanding of these multi-modular receptors and how these individual components confer particular properties to CAR-Ts. In addition, we will discuss extrinsic factors that can be manipulated to influence CAR-T performance such as choice of cellular population, culturing conditions and additional modifications that enhance their activity particularly in solid tumors. Finally, we will also consider the emerging toxicity associated with CAR-Ts. By breaking apart the CAR and examining the role of each piece, we can build a better functioning cellular vehicle for optimized treatment of cancer patients. PMID:26797495

  12. An infectious bat chimeric influenza virus harboring the entry machinery of a influenza A virus

    PubMed Central

    Juozapaitis, Mindaugas; Moreira, Étori Aguiar; Mena, Ignacio; Giese, Sebastian; Riegger, David; Pohlmann, Anne; Höper, Dirk; Zimmer, Gert; Beer, Martin; García-Sastre, Adolfo; Schwemmle, Martin

    2017-01-01

    In 2012 the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the HA and NA proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event. PMID:25055345

  13. Suppression of the biosynthesis of proanthocyanidin in Arabidopsis by a chimeric PAP1 repressor.

    PubMed

    Matsui, Kyoko; Tanaka, Hideo; Ohme-Takagi, Masaru

    2004-11-01

    Flavonoids are secondary metabolites that are specific to higher plants. PAP1, a member of the family of MYB domain transcription factors in Arabidopsis, is a positive regulator of the biosynthesis of anthocyanin. We show here that a chimeric PAP1 repressor, in which the EAR-motif repression domain from SUPERMAN was fused to PAP1, suppressed the expression of four flavonoid biosynthetic genes, namely CHS, DFR, LDOX, and BAN, in siliques, and inhibited the accumulation of proanthocyanidin, even in the presence of an endogenous positive regulator, such as TT2. This suppression resulted in the formation of light yellow seeds in 35S::PAP1SRDX transgenic plants. Our results indicate that PAP1 has the potential ability to regulate the biosynthesis not only of anthocyanin but also of proanthocyanidin. Our gene silencing system, using chimeric repressors, appears to be a useful tool for the manipulation of the biosynthesis of secondary metabolites in plants.

  14. Homology among divergent Paleozoic tetrapod clades.

    PubMed

    Carroll, R L

    1999-01-01

    A stringent definition of homology is necessary to establish phylogenetic relationships among Paleozoic amphibians. Many derived characters exhibited by divergent clades of Carboniferous lepospondyls resemble those achieved convergently among Cenozoic squamates that have elongate bodies and reduced limbs, and by lineages of modern amphibians that have undergone miniaturization. Incongruent character distribution, poorly resolved cladograms and functionally improbable character transformations determined by phylogenetic analysis suggest that convergence was also common among Paleozoic amphibians with a skull length under 3 cm, including lepospondyls, early amniotes and the putative ancestors of modern amphibians. For this reason, it is injudicious to equate apparent synapomorphy (perceived common presence of a particular derived character in two putative sister-taxa) with strict homology of phylogenetic origin. Identification of homology by the similarity of structure, anatomical position and pattern of development is insufficient to establish the synapomorphy of bone and limb loss or precocial ossification of vertebral centra, which are common among small Paleozoic amphibians. The only way in which synapomorphies can be established definitively is through the discovery and recognition of the trait in question in basal members of each of the clades under study, and in their immediate common ancestors.

  15. Faster sequence homology searches by clustering subsequences

    PubMed Central

    Suzuki, Shuji; Kakuta, Masanori; Ishida, Takashi; Akiyama, Yutaka

    2015-01-01

    Motivation: Sequence homology searches are used in various fields. New sequencing technologies produce huge amounts of sequence data, which continuously increase the size of sequence databases. As a result, homology searches require large amounts of computational time, especially for metagenomic analysis. Results: We developed a fast homology search method based on database subsequence clustering, and implemented it as GHOSTZ. This method clusters similar subsequences from a database to perform an efficient seed search and ungapped extension by reducing alignment candidates based on triangle inequality. The database subsequence clustering technique achieved an ∼2-fold increase in speed without a large decrease in search sensitivity. When we measured with metagenomic data, GHOSTZ is ∼2.2–2.8 times faster than RAPSearch and is ∼185–261 times faster than BLASTX. Availability and implementation: The source code is freely available for download at http://www.bi.cs.titech.ac.jp/ghostz/ Contact: akiyama@cs.titech.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25432166

  16. Weak homological dimensions and biflat Koethe algebras

    SciTech Connect

    Pirkovskii, A Yu

    2008-06-30

    The homological properties of metrizable Koethe algebras {lambda}(P) are studied. A criterion for an algebra A={lambda}(P) to be biflat in terms of the Koethe set P is obtained, which implies, in particular, that for such algebras the properties of being biprojective, biflat, and flat on the left are equivalent to the surjectivity of the multiplication operator A otimes-hat A{yields}A. The weak homological dimensions (the weak global dimension w.dg and the weak bidimension w.db) of biflat Koethe algebras are calculated. Namely, it is shown that the conditions w.db {lambda}(P)<=1 and w.dg {lambda}(P)<=1 are equivalent to the nuclearity of {lambda}(P); and if {lambda}(P) is non-nuclear, then w.dg {lambda}(P)=w.db {lambda}(P)=2. It is established that the nuclearity of a biflat Koethe algebra {lambda}(P), under certain additional conditions on the Koethe set P, implies the stronger estimate db {lambda}(P), where db is the (projective) bidimension. On the other hand, an example is constructed of a nuclear biflat Koethe algebra {lambda}(P) such that db {lambda}(P)=2 (while w.db {lambda}(P)=1). Finally, it is shown that many biflat Koethe algebras, while not being amenable, have trivial Hochschild homology groups in positive degrees (with arbitrary coefficients). Bibliography: 37 titles.

  17. PubServer: literature searches by homology

    PubMed Central

    Jaroszewski, Lukasz; Koska, Laszlo; Sedova, Mayya; Godzik, Adam

    2014-01-01

    PubServer, available at http://pubserver.burnham.org/, is a tool to automatically collect, filter and analyze publications associated with groups of homologous proteins. Protein entries in databases such as Entrez Protein database at NCBI contain information about publications associated with a given protein. The scope of these publications varies a lot: they include studies focused on biochemical functions of individual proteins, but also reports from genome sequencing projects that introduce tens of thousands of proteins. Collecting and analyzing publications related to sets of homologous proteins help in functional annotation of novel protein families and in improving annotations of well-studied protein families or individual genes. However, performing such collection and analysis manually is a tedious and time-consuming process. PubServer automatically collects identifiers of homologous proteins using PSI-Blast, retrieves literature references from corresponding database entries and filters out publications unlikely to contain useful information about individual proteins. It also prepares simple vocabulary statistics from titles, abstracts and MeSH terms to identify the most frequently occurring keywords, which may help to quickly identify common themes in these publications. The filtering criteria applied to collected publications are user-adjustable. The results of the server are presented as an interactive page that allows re-filtering and different presentations of the output. PMID:24957597

  18. Dental homologies in lamniform sharks (Chondrichthyes: Elasmobranchii).

    PubMed

    Shimada, Kenshu

    2002-01-01

    The dentitions of lamniform sharks are said to exhibit a unique heterodonty called the "lamnoid tooth pattern." The presence of an inflated hollow "dental bulla" on each jaw cartilage allows the recognition of homologous teeth across most modern macrophagous lamniforms based on topographic correspondence through the "similarity test." In most macrophagous lamniforms, three tooth rows are supported by the upper dental bulla: two rows of large anterior teeth followed by a row of small intermediate teeth. The lower tooth row occluding between the two rows of upper anterior teeth is the first lower anterior tooth row. Like the first and second lower anterior tooth rows, the third lower tooth row is supported by the dental bulla and may be called the first lower intermediate tooth row. The lower intermediate tooth row occludes between the first and second upper lateral tooth rows situated distal to the upper dental bulla, and the rest of the upper and lower tooth rows, all called lateral tooth rows, occlude alternately. Tooth symmetry cannot be used to identify their dental homology. The presence of dental bullae can be regarded as a synapomorphy of Lamniformes and this character is more definable than the "lamnoid tooth pattern." The formation of the tooth pattern appears to be related to the evolution of dental bullae. This study constitutes the first demonstration of supraspecific tooth-to-tooth dental homologies in nonmammalian vertebrates. Copyright 2002 Wiley-Liss, Inc.

  19. PubServer: literature searches by homology.

    PubMed

    Jaroszewski, Lukasz; Koska, Laszlo; Sedova, Mayya; Godzik, Adam

    2014-07-01

    PubServer, available at http://pubserver.burnham.org/, is a tool to automatically collect, filter and analyze publications associated with groups of homologous proteins. Protein entries in databases such as Entrez Protein database at NCBI contain information about publications associated with a given protein. The scope of these publications varies a lot: they include studies focused on biochemical functions of individual proteins, but also reports from genome sequencing projects that introduce tens of thousands of proteins. Collecting and analyzing publications related to sets of homologous proteins help in functional annotation of novel protein families and in improving annotations of well-studied protein families or individual genes. However, performing such collection and analysis manually is a tedious and time-consuming process. PubServer automatically collects identifiers of homologous proteins using PSI-Blast, retrieves literature references from corresponding database entries and filters out publications unlikely to contain useful information about individual proteins. It also prepares simple vocabulary statistics from titles, abstracts and MeSH terms to identify the most frequently occurring keywords, which may help to quickly identify common themes in these publications. The filtering criteria applied to collected publications are user-adjustable. The results of the server are presented as an interactive page that allows re-filtering and different presentations of the output.

  20. Advances in Homology Protein Structure Modeling

    PubMed Central

    Xiang, Zhexin

    2007-01-01

    Homology modeling plays a central role in determining protein structure in the structural genomics project. The importance of homology modeling has been steadily increasing because of the large gap that exists between the overwhelming number of available protein sequences and experimentally solved protein structures, and also, more importantly, because of the increasing reliability and accuracy of the method. In fact, a protein sequence with over 30% identity to a known structure can often be predicted with an accuracy equivalent to a low-resolution X-ray structure. The recent advances in homology modeling, especially in detecting distant homologues, aligning sequences with template structures, modeling of loops and side chains, as well as detecting errors in a model, have contributed to reliable prediction of protein structure, which was not possible even several years ago. The ongoing efforts in solving protein structures, which can be time-consuming and often difficult, will continue to spur the development of a host of new computational methods that can fill in the gap and further contribute to understanding the relationship between protein structure and function. PMID:16787261

  1. Mismatch repair during homologous and homeologous recombination.

    PubMed

    Spies, Maria; Fishel, Richard

    2015-03-02

    Homologous recombination (HR) and mismatch repair (MMR) are inextricably linked. HR pairs homologous chromosomes before meiosis I and is ultimately responsible for generating genetic diversity during sexual reproduction. HR is initiated in meiosis by numerous programmed DNA double-strand breaks (DSBs; several hundred in mammals). A characteristic feature of HR is the exchange of DNA strands, which results in the formation of heteroduplex DNA. Mismatched nucleotides arise in heteroduplex DNA because the participating parental chromosomes contain nonidentical sequences. These mismatched nucleotides may be processed by MMR, resulting in nonreciprocal exchange of genetic information (gene conversion). MMR and HR also play prominent roles in mitotic cells during genome duplication; MMR rectifies polymerase misincorporation errors, whereas HR contributes to replication fork maintenance, as well as the repair of spontaneous DSBs and genotoxic lesions that affect both DNA strands. MMR suppresses HR when the heteroduplex DNA contains excessive mismatched nucleotides, termed homeologous recombination. The regulation of homeologous recombination by MMR ensures the accuracy of DSB repair and significantly contributes to species barriers during sexual reproduction. This review discusses the history, genetics, biochemistry, biophysics, and the current state of studies on the role of MMR in homologous and homeologous recombination from bacteria to humans.

  2. Homologous recombination using bacterial artificial chromosomes.

    PubMed

    Lai, Cary; Fischer, Tobias; Munroe, Elizabeth

    2015-02-02

    This protocol introduces the technique of homologous recombination in bacteria to insert a linear DNA fragment into bacterial artificial chromosomes (BACs). Homologous recombination allows the modification of large DNA molecules, in contrast with conventional restriction endonuclease-based strategies, which cleave large DNAs into numerous fragments and are unlikely to permit the precise targeting afforded by recombination-based approaches. The method uses a phage lambda-derived recombination system (using exo, beta, and gam) as well as other enzymatic activities provided by the host (Escherichia coli). In the method described here, a DNA fragment encoding enhanced cyan fluorescent protein is inserted immediately after the start codon of the gene encoding choline acetyltransferase ("ChAT"), the final enzyme in acetylcholine biosynthesis, using homologous recombination between sequences that are present both on the introduced DNA fragment and in the target BAC. The desired recombination products are identified via positive selection for resistance to kanamycin. In principle, the resulting modified BAC could be used to produce transgenic mice that express this fluorescent protein in cholinergic neurons. The approach described here could be used to insert any DNA fragment.

  3. Mismatch Repair during Homologous and Homeologous Recombination

    PubMed Central

    Spies, Maria; Fishel, Richard

    2015-01-01

    Homologous recombination (HR) and mismatch repair (MMR) are inextricably linked. HR pairs homologous chromosomes before meiosis I and is ultimately responsible for generating genetic diversity during sexual reproduction. HR is initiated in meiosis by numerous programmed DNA double-strand breaks (DSBs; several hundred in mammals). A characteristic feature of HR is the exchange of DNA strands, which results in the formation of heteroduplex DNA. Mismatched nucleotides arise in heteroduplex DNA because the participating parental chromosomes contain nonidentical sequences. These mismatched nucleotides may be processed by MMR, resulting in nonreciprocal exchange of genetic information (gene conversion). MMR and HR also play prominent roles in mitotic cells during genome duplication; MMR rectifies polymerase misincorporation errors, whereas HR contributes to replication fork maintenance, as well as the repair of spontaneous DSBs and genotoxic lesions that affect both DNA strands. MMR suppresses HR when the heteroduplex DNA contains excessive mismatched nucleotides, termed homeologous recombination. The regulation of homeologous recombination by MMR ensures the accuracy of DSB repair and significantly contributes to species barriers during sexual reproduction. This review discusses the history, genetics, biochemistry, biophysics, and the current state of studies on the role of MMR in homologous and homeologous recombination from bacteria to humans. PMID:25731766

  4. Chimeric virus-like particles containing influenza HA antigen and GPI-CCL28 induce long-lasting mucosal immunity against H3N2 viruses

    PubMed Central

    Mohan, Teena; Berman, Zachary; Luo, Yuan; Wang, Chao; Wang, Shelly; Compans, Richard W.; Wang, Bao-Zhong

    2017-01-01

    Influenza virus is a significant cause of morbidity and mortality, with worldwide seasonal epidemics. The duration and quality of humoral immunity and generation of immunological memory to vaccines is critical for protective immunity. In the current study, we examined the long-lasting protective efficacy of chimeric VLPs (cVLPs) containing influenza HA and GPI-anchored CCL28 as antigen and mucosal adjuvant, respectively, when immunized intranasally in mice. We report that the cVLPs induced significantly higher and sustainable levels of virus-specific antibody responses, especially IgA levels and hemagglutination inhibition (HAI) titers, more than 8-month post-vaccination compared to influenza VLPs without CCL28 or influenza VLPs physically mixed with sCCL28 (soluble) in mice. After challenging the vaccinated animals at month 8 with H3N2 viruses, the cVLP group also demonstrated strong recall responses. On day 4 post-challenge, we measured increased antibody levels, ASCs and HAI titers with reduced viral load and inflammatory responses in the cVLP group. The animals vaccinated with the cVLP showed 20% cross-protection against drifted (Philippines) and 60% protection against homologous (Aichi) H3N2 viruses. Thus, the results suggest that the GPI-anchored CCL28 induces significantly higher mucosal antibody responses, involved in providing long-term cross-protection against H3N2 influenza virus when compared to other vaccination groups. PMID:28067290

  5. Enhanced efficacy of an AAV vector encoding chimeric, highly secreted acid alpha-glucosidase in glycogen storage disease type II.

    PubMed

    Sun, Baodong; Zhang, Haoyue; Benjamin, Daniel K; Brown, Talmage; Bird, Andrew; Young, Sarah P; McVie-Wylie, Alison; Chen, Y-T; Koeberl, Dwight D

    2006-12-01

    Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) is an inherited muscular dystrophy caused by deficiency in the activity of the lysosomal enzyme acid alpha-glucosidase (GAA). We hypothesized that chimeric GAA containing an alternative signal peptide could increase the secretion of GAA from transduced cells and enhance the receptor-mediated uptake of GAA in striated muscle. The relative secretion of chimeric GAA from transfected 293 cells increased up to 26-fold. Receptor-mediated uptake of secreted, chimeric GAA corrected cultured GSD-II patient cells. High-level hGAA was sustained in the plasma of GSD-II mice for 24 weeks following administration of an AAV2/8 vector encoding chimeric GAA; furthermore, GAA activity was increased and glycogen content was significantly reduced in striated muscle and in the brain. Administration of only 1 x 10(10) vector particles increased GAA activity in the heart and diaphragm for >18 weeks, whereas 3 x 10(10) vector particles increased GAA activity and reduced glycogen content in the heart, diaphragm, and quadriceps. Furthermore, an AAV2/2 vector encoding chimeric GAA produced secreted hGAA for >12 weeks in the majority of treated GSD-II mice. Thus, chimeric, highly secreted GAA enhanced the efficacy of AAV vector-mediated gene therapy in GSD-II mice.

  6. Designing a recombinant chimeric construct contain MUC1 and HER2 extracellular domain for prediagnostic breast cancer.

    PubMed

    Gheybi, Elaheh; Amani, Jafar; Salmanian, Ali Hatef; Mashayekhi, Farhad; Khodi, Samaneh

    2014-11-01

    Breast cancer is the most common cancer among women in the world. One of the approaches for diagnosis of breast cancer is detection of its tumor-associated markers. Mucin 1 (MUC1), a tumor-associated antigen, is a transmembrane glycoprotein expressed by normal epithelial cells and overexpressed by carcinomas of epithelial origin. Also, human epidermal growth factor receptor-2 (HER2/erbB-2) belongs to the one of four members of tyrosin kinase type 1 family in which overexpression of HER2 is associated with malignancy in breast cancer. This study was aimed to bioinformatics analysis and designing a recombinant chimeric protein containing MUC1 and HER2 antigens to express in prokaryotic host (Escherichia coli) as breast cancer diagnosis tools. The immunogenic sequences of MUC1 and HER2 were extracted and fused together by a linker. The chimeric construct was analyzed by bioinformatics softwares. The optimization and purification, evaluation of the expression of chimeric protein was performed using Western blotting, ELISA, and immunohistochemistry. The results showed that the chimeric construct was stable and immunogenic domains were exposed. The pET-28a vector containing chimeric gene had high level of protein expression. The recombinant chimeric protein was confirmed using Western blotting, and it was investigated using ELISA and IHC. Then, the MUC1 and HER2 combined peptides can be used as coating antigens in ELISA for detection of antibodies against MUC1 or HER2 in human serum.

  7. Humanized neuronal chimeric mouse brain generated by neonatally engrafted human iPSC-derived primitive neural progenitor cells

    PubMed Central

    Chen, Chen

    2016-01-01

    The creation of a humanized chimeric mouse nervous system permits the study of human neural development and disease pathogenesis using human cells in vivo. Humanized glial chimeric mice with the brain and spinal cord being colonized by human glial cells have been successfully generated. However, generation of humanized chimeric mouse brains repopulated by human neurons to possess a high degree of chimerism have not been well studied. Here we created humanized neuronal chimeric mouse brains by neonatally engrafting the distinct and highly neurogenic human induced pluripotent stem cell (hiPSC)–derived rosette-type primitive neural progenitors. These neural progenitors predominantly differentiate to neurons, which disperse widely throughout the mouse brain with infiltration of the cerebral cortex and hippocampus at 6 and 13 months after transplantation. Building upon the hiPSC technology, we propose that this potentially unique humanized neuronal chimeric mouse model will provide profound opportunities to define the structure, function, and plasticity of neural networks containing human neurons derived from a broad variety of neurological disorders. PMID:27882348

  8. Transcriptome analysis revealed chimeric RNAs, single nucleotide polymorphisms and allele-specific expression in porcine prenatal skeletal muscle

    PubMed Central

    Yang, Yalan; Tang, Zhonglin; Fan, Xinhao; Xu, Kui; Mu, Yulian; Zhou, Rong; Li, Kui

    2016-01-01

    Prenatal skeletal muscle development genetically determines postnatal muscle characteristics such as growth and meat quality in pigs. However, the molecular mechanisms underlying prenatal skeletal muscle development remain unclear. Here, we performed the first genome-wide analysis of chimeric RNAs, single nuclear polymorphisms (SNPs) and allele-specific expression (ASE) in prenatal skeletal muscle in pigs. We identified 14,810 protein coding genes and 163 high-confidence chimeric RNAs expressed in prenatal skeletal muscle. More than 94.5% of the chimeric RNAs obeyed the canonical GT/AG splice rule and were trans-splicing events. Ten and two RNAs were aligned to human and mouse chimeric transcripts, respectively. We detected 106,457 high-quality SNPs (6,955 novel), which were mostly (89.09%) located within QTLs for production traits. The high proportion of non-exonic SNPs revealed the incomplete annotation status of the current swine reference genome. ASE analysis revealed that 11,300 heterozygous SNPs showed allelic imbalance, whereas 131 ASE variants were located in the chimeric RNAs. Moreover, 4 ASE variants were associated with various economically relevant traits of pigs. Taken together, our data provide a source for studies of chimeric RNAs and biomarkers for pig breeding, while illuminating the complex transcriptional events underlying prenatal skeletal muscle development in mammals. PMID:27352850

  9. Current status of prediction of drug disposition and toxicity in humans using chimeric mice with humanized liver.

    PubMed

    Kitamura, Shigeyuki; Sugihara, Kazumi

    2014-01-01

    1. Human-chimeric mice with humanized liver have been constructed by transplantation of human hepatocytes into several types of mice having genetic modifications that injure endogenous liver cells. Here, we focus on liver urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice, which are the most widely used human-chimeric mice. Studies so far indicate that drug metabolism, drug transport, pharmacological effects and toxicological action in these mice are broadly similar to those in humans. 2. Expression of various drug-metabolizing enzymes is known to be different between humans and rodents. However, the expression pattern of cytochrome P450, aldehyde oxidase and phase II enzymes in the liver of human-chimeric mice resembles that in humans, not that in the host mice. 3. Metabolism of various drugs, including S-warfarin, zaleplon, ibuprofen, naproxen, coumarin, troglitazone and midazolam, in human-chimeric mice is mediated by human drug-metabolizing enzymes, not by host mouse enzymes, and thus resembles that in humans. 4. Pharmacological and toxicological effects of various drugs in human-chimeric mice are also similar to those in humans. 5. The current consensus is that chimeric mice with humanized liver are useful to predict drug metabolism catalyzed by cytochrome P450, aldehyde oxidase and phase II enzymes in humans in vivo and in vitro. Some remaining issues are discussed in this review.

  10. Pharmacokinetics and effects on serum cholinesterase activities of organophosphorus pesticides acephate and chlorpyrifos in chimeric mice transplanted with human hepatocytes.

    PubMed

    Suemizu, Hiroshi; Sota, Shigeto; Kuronuma, Miyuki; Shimizu, Makiko; Yamazaki, Hiroshi

    2014-11-01

    Organophosphorus pesticides acephate and chlorpyrifos in foods have potential to impact human health. The aim of the current study was to investigate the pharmacokinetics of acephate and chlorpyrifos orally administered at lowest-observed-adverse-effect-level doses in chimeric mice transplanted with human hepatocytes. Absorbed acephate and its metabolite methamidophos were detected in serum from wild type mice and chimeric mice orally administered 150mg/kg. Approximately 70% inhibition of cholinesterase was evident in plasma of chimeric mice with humanized liver (which have higher serum cholinesterase activities than wild type mice) 1day after oral administrations of acephate. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated plasma concentrations of acephate and chlorpyrifos in humans were consistent with reported concentrations. Acephate cleared similarly in humans and chimeric mice but accidental/incidental overdose levels of chlorpyrifos cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in mice. The data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential of organophosphorus pesticides. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Homology and ontogeny: pattern and process in comparative developmental biology.

    PubMed

    Scholtz, Gerhard

    2005-11-01

    In this article the interface between development and homology is discussed. Development is here interpreted as a sequence of evolutionarily independent stages. Any approach stressing the importance of specific developmental stages is rejected. A homology definition is favoured which includes similarity, and complexity serves as a test for homology. Complexity is seen as the possibility of subdividing a character into evolutionarily independent corresponding substructures. Topology as a test for homology is critically discussed because corresponding positions are not necessarily indicative of homology. Complexity can be used twofold for homology assessments of development: either stages or processes of development are homologized. These two approaches must not be con-flated. This distinction leads to the conclusion that there is no ontogenetic homology "criterion".

  12. Lessons learned from a highly-active CD22-specific chimeric antigen receptor

    PubMed Central

    Long, Adrienne H.; Haso, Waleed M.; Orentas, Rimas J.

    2013-01-01

    CD22 is an attractive target for the development of immunotherapeutic approaches for the therapy of B-cell malignancies. In particular, an m971 antibody-derived, second generation chimeric antigen receptor (CAR) that targets CD22 holds significant therapeutic promise. The key aspect for the development of such a highly-active CAR was its ability to target a membrane-proximal epitope of CD22. PMID:23734316

  13. Lessons learned from a highly-active CD22-specific chimeric antigen receptor.

    PubMed

    Long, Adrienne H; Haso, Waleed M; Orentas, Rimas J

    2013-04-01

    CD22 is an attractive target for the development of immunotherapeutic approaches for the therapy of B-cell malignancies. In particular, an m971 antibody-derived, second generation chimeric antigen receptor (CAR) that targets CD22 holds significant therapeutic promise. The key aspect for the development of such a highly-active CAR was its ability to target a membrane-proximal epitope of CD22.

  14. Chimeric mice with a humanized liver as an animal model of troglitazone-induced liver injury.

    PubMed

    Kakuni, Masakazu; Morita, Mayu; Matsuo, Kentaro; Katoh, Yumiko; Nakajima, Miki; Tateno, Chise; Yokoi, Tsuyoshi

    2012-10-02

    Troglitazone (Tro) is a thiazolidinedione antidiabetic drug that was withdrawn from the market due to its association with idiosyncratic severe liver injury. Tro has never induced liver injury in experimental animals in vivo. It was assumed that the species differences between human and experimental animals in the pharmaco- or toxicokinetics of Tro might be associated with these observations. In this study, we investigated whether a chimeric mouse with a humanized liver that we previously established, whose replacement index with human hepatocytes is up to 92% can reproduce Tro-induced liver injury. When the chimeric mice were orally administered Tro for 14 or 23 days (1000mg/kg/day), serum alanine aminotransferase (ALT) was significantly increased by 2.1- and 3.6-fold, respectively. Co-administration of l-buthionine sulfoximine (10mM in drinking water), an inhibitor of glutathione (GSH) synthesis, unexpectedly prevented the Tro-dependent increase of ALT, which suggests that the GSH scavenging pathway will not be involved in Tro-induced liver injury. To elucidate the mechanism of the onset of liver injury, hepatic GSH content, the level of oxidative stress markers and phase I and phase II drug metabolizing enzymes were determined. However, these factors were not associated with Tro-induced liver injury. An immune-mediated reaction may be associated with Tro-induced liver toxicity in vivo, because the chimeric mouse is derived from an immunodeficient SCID mouse. In conclusion, we successfully reproduced Tro-induced liver injury using chimeric mice with a humanized liver, which provides a new animal model for studying idiosyncratic drug-induced liver injury. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  15. Transient Expression of Chimeric Genes Delivered into Pollen by Microprojectile Bombardment 1

    PubMed Central

    Twell, David; Klein, Theodore M.; Fromm, Michael E.; McCormick, Sheila

    1989-01-01

    Chimeric genes containing a pollen-specific promoter from tomato (Lycopersicon esculentum) or the CaMV35S promoter were transiently expressed following their introduction into tobacco (Nicotiana tabacum) pollen using high velocity microprojectiles. Transient expression of the microprojectile-introduced genes in leaves and pollen was similar to that observed for these genes in stably transformed tobacco plants. Images Figure 2 PMID:16667175

  16. Conformational influence of the ribose 2'-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes

    NASA Technical Reports Server (NTRS)

    Egli, M.; Usman, N.; Rich, A.

    1993-01-01

    We have crystallized three double-helical DNA-RNA chimeric duplexes and determined their structures by X-ray crystallography at resolutions between 2 and 2.25 A. The two self-complementary duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC).r(GCG)d(TATACCC), were found to adopt A-type conformations. The crystal structures are non-isomorphous, and the crystallographic environments for the three chimeras are different. A number of intramolecular interactions of the ribose 2'-hydroxyl groups contribute to the stabilization of the A-conformation. Hydrogen bonds between 2'-hydroxyls and 5'-oxygens or phosphate oxygens, in addition to the previously observed hydrogen bonds to 1'-oxygens of adjacent riboses and deoxyriboses, are observed in the DNA-RNA chimeric duplexes. The crystalline chimeric duplexes do not show a transition between the DNA A- and B-conformations. CD spectra suggest that the Okazaki fragment assumes an A-conformation in solution as well. In this molecule the three RNA residues may therefore lock the complete decamer in the A-conformation. Crystals of an all-DNA strand with the same sequence as the self-complementary chimeras show a morphology which is different from those of the chimera crystals. Moreover, the oligonucleotide does not match any of the sequence characteristics of DNAs usually adopting the A-conformation in the crystalline state (e.g., octamers with short alternating stretches of purines and pyrimidines). In DNA-RNA chimeric duplexes, it is therefore possible that a single RNA residue can drive the conformational equilibrium toward the A-conformation.

  17. Cytokine Release Syndrome After Chimeric Antigen Receptor T Cell Therapy for Acute Lymphoblastic Leukemia.

    PubMed

    Fitzgerald, Julie C; Weiss, Scott L; Maude, Shannon L; Barrett, David M; Lacey, Simon F; Melenhorst, J Joseph; Shaw, Pamela; Berg, Robert A; June, Carl H; Porter, David L; Frey, Noelle V; Grupp, Stephan A; Teachey, David T

    2017-02-01

    Initial success with chimeric antigen receptor-modified T cell therapy for relapsed/refractory acute lymphoblastic leukemia is leading to expanded use through multicenter trials. Cytokine release syndrome, the most severe toxicity, presents a novel critical illness syndrome with limited data regarding diagnosis, prognosis, and therapy. We sought to characterize the timing, severity, and intensive care management of cytokine release syndrome after chimeric antigen receptor-modified T cell therapy. Retrospective cohort study. Academic children's hospital. Thirty-nine subjects with relapsed/refractory acute lymphoblastic leukemia treated with chimeric antigen receptor-modified T cell therapy on a phase I/IIa clinical trial (ClinicalTrials.gov number NCT01626495). All subjects received chimeric antigen receptor-modified T cell therapy. Thirteen subjects with cardiovascular dysfunction were treated with the interleukin-6 receptor antibody tocilizumab. Eighteen subjects (46%) developed grade 3-4 cytokine release syndrome, with prolonged fever (median, 6.5 d), hyperferritinemia (median peak ferritin, 60,214 ng/mL), and organ dysfunction. Fourteen (36%) developed cardiovascular dysfunction treated with vasoactive infusions a median of 5 days after T cell therapy. Six (15%) developed acute respiratory failure treated with invasive mechanical ventilation a median of 6 days after T cell therapy; five met criteria for acute respiratory distress syndrome. Encephalopathy, hepatic, and renal dysfunction manifested later than cardiovascular and respiratory dysfunction. Subjects had a median of 15 organ dysfunction days (interquartile range, 8-20). Treatment with tocilizumab in 13 subjects resulted in rapid defervescence (median, 4 hr) and clinical improvement. Grade 3-4 cytokine release syndrome occurred in 46% of patients following T cell therapy for relapsed/refractory acute lymphoblastic leukemia. Clinicians should be aware of expanding use of this breakthrough therapy and

  18. Production and Characterisation of a Neutralising Chimeric Antibody against Botulinum Neurotoxin A

    PubMed Central

    Prigent, Julie; Mazuet, Christelle; Boquet, Didier; Lamourette, Patricia; Volland, Hervé; Popoff, Michel R.; Créminon, Christophe; Simon, Stéphanie

    2010-01-01

    Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A) is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC) as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains). Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14) and insect cells (Sf9). After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM) and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species. PMID:20967241

  19. Generation of cloned and chimeric embryos/offspring using the new methods of animal biotechnology.

    PubMed

    Skrzyszowska, Maria; Karasiewicz, Jolanta; Bednarczyk, Marek; Samiec, Marcin; Smorag, Zdzisław; Waś, Bogusław; Guszkiewicz, Andrzej; Korwin-Kossakowski, Maciej; Górniewska, Maria; Szablisty, Ewa; Modliński, Jacek A; Łakota, Paweł; Wawrzyńska, Magdalena; Sechman, Andrzej; Wojtysiak, Dorota; Hrabia, Anna; Mika, Maria; Lisowski, Mirosław; Czekalski, Przemysław; Rzasa, Janusz; Kapkowska, Ewa

    2006-01-01

    The article summarizes results of studies concerning: 1/ qualitative evaluation of pig nuclear donor cells to somatic cell cloning, 2/ developmental potency of sheep somatic cells to create chimera, 3/ efficient production of chicken chimera. The quality of nuclear donor cells is one of the most important factors to determine the efficiency of somatic cell cloning. Morphological criteria commonly used for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Therefore, different types of somatic cells being the source of genomic DNA in the cloning procedure were analyzed on apoptosis with the use of live-DNA or plasma membrane fluorescent markers. It has been found that morphological criteria are a sufficient selection factor for qualitative evaluation of nuclear donor cells to somatic cell cloning. Developmental potencies of sheep somatic cells in embryos and chimeric animals were studied using blastocyst complementation test. Fetal fibroblasts stained with vital fluorescent dye and microsurgically placed in morulae or blastocysts were later identified in embryos cultured in vitro. Transfer of Polish merino blastocysts harbouring Heatherhead fibroblasts to recipient ewes brought about normal births at term. Newly-born animals were of merino appearance with dark patches on their noses, near the mouth and on their clovens. This overt chimerism shows that fetal fibroblasts introduced to sheep morulae/blastocysts revealed full developmental plasticity. To achieve the efficient production of chicken chimeras, the blastodermal cells from embryos of the donor breeds, (Green-legged Partridgelike breed or GPxAraucana) were transferred into the embryos of the recipient breed (White Leghorn), and the effect of chimerism on the selected reproductive and physiological traits of recipients was examined. Using the model which allowed identification of the chimerism at many loci, it has been found that 93.9% of the examined birds

  20. High male chimerism in the female breast shows quantitative links with cancer.

    PubMed

    Dhimolea, Eugen; Denes, Viktoria; Lakk, Monika; Al-Bazzaz, Sana; Aziz-Zaman, Sonya; Pilichowska, Monika; Geck, Peter

    2013-08-15

    Clinical observations suggest that pregnancy provides protection against cancer. The mechanisms involved, however, remain unclear. Fetal cells are known to enter the mother's circulation during pregnancy and establish microchimerism. We investigated if pregnancy-related embryonic/fetal stem cell integration plays a role in breast cancer. A high-sensitivity Y-chromosome assay was developed to trace male allogeneic cells (from male fetus) in females. Fixed-embedded samples (n = 206) from both normal and breast cancer patients were screened for microchimerism. The results were combined with matching clinicopathological and histological parameters and processed statistically. The results show that in our samples (182 informative) more than half of healthy women (56%) carried male cells in their breast tissue for decades (n = 68), while only one out of five in the cancer sample pool (21%) (n = 114) (odds ratio = 4.75, CI at 95% 2.34-9.69; p = 0.0001). The data support the notion that a biological link may exist between chimerism and tissue-integrity. The correlation, however, is non-linear, since male microchimerism in excess ("hyperchimerism") is also involved in cancer. The data suggest a link between hyperchimerism and HER2-type cancers, while decreased chimerism ("hypochimerism") associates with ER/PR-positive (luminal-type) breast cancers. Chimerism levels that correlate with protection appear to be non-random and share densities with the mammary progenitor components of the stem cell lineage in the breast. The results suggest that protection may involve stem/progenitor level interactions and implicate novel quantitative mechanisms in chimerism biology. Copyright © 2013 UICC.

  1. High-resolution air quality simulation over Europe with the chemistry transport model CHIMERE

    NASA Astrophysics Data System (ADS)

    Terrenoire, E.; Bessagnet, B.; Rouïl, L.; Tognet, F.; Pirovano, G.; Létinois, L.; Beauchamp, M.; Colette, A.; Thunis, P.; Amann, M.; Menut, L.

    2015-01-01

    A modified version of CHIMERE 2009, including new methodologies in emissions modelling and an urban correction, is used to perform a simulation at high resolution (0.125° × 0.0625°) over Europe for the year 2009. The model reproduces the temporal variability of NO2, O3, PM10, PM2.5 better at rural (RB) than urban (UB) background stations, with yearly correlation values for the different pollutants ranging between 0.62 and 0.77 at RB sites and between 0.52 and 0.73 at UB sites. Also, the fractional biases (FBs) show that the model performs slightly better at RB sites than at UB sites for NO2 (RB = -33.9%, UB = -53.6%), O3 (RB = 20.1%, UB = 25.2%) and PM10 (RB = -5.50%, UB = -20.1%). The difficulties for the model in reproducing NO2 concentrations can be attributed to the general underestimation of NOx emissions as well as to the adopted horizontal resolution, which represents only partially the spatial gradient of the emissions over medium-size and small cities. The overestimation of O3 by the model is related to the NO2 underestimation and the overestimated O3 concentrations of the lateral boundary conditions. At UB sites, CHIMERE reproduces PM2.5 better than PM10. This is primarily the result of an underestimation of coarse particulate matter (PM) associated with uncertainties in secondary organic aerosol (SOA) chemistry and its precursor emissions (Po valley and Mediterranean basin), dust (south of Spain) and sea salt (western Europe). The results suggest that future work should focus on the development of national bottom-up emission inventories including a better account for semi-volatile organic compounds and their conversion to SOA, the improvement of the CHIMERE urban parameterization, the introduction into CHIMERE of the coarse nitrate chemistry and an advanced parameterization accounting for windblown dust emissions.

  2. Conformational influence of the ribose 2'-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes

    NASA Technical Reports Server (NTRS)

    Egli, M.; Usman, N.; Rich, A.

    1993-01-01

    We have crystallized three double-helical DNA-RNA chimeric duplexes and determined their structures by X-ray crystallography at resolutions between 2 and 2.25 A. The two self-complementary duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC).r(GCG)d(TATACCC), were found to adopt A-type conformations. The crystal structures are non-isomorphous, and the crystallographic environments for the three chimeras are different. A number of intramolecular interactions of the ribose 2'-hydroxyl groups contribute to the stabilization of the A-conformation. Hydrogen bonds between 2'-hydroxyls and 5'-oxygens or phosphate oxygens, in addition to the previously observed hydrogen bonds to 1'-oxygens of adjacent riboses and deoxyriboses, are observed in the DNA-RNA chimeric duplexes. The crystalline chimeric duplexes do not show a transition between the DNA A- and B-conformations. CD spectra suggest that the Okazaki fragment assumes an A-conformation in solution as well. In this molecule the three RNA residues may therefore lock the complete decamer in the A-conformation. Crystals of an all-DNA strand with the same sequence as the self-complementary chimeras show a morphology which is different from those of the chimera crystals. Moreover, the oligonucleotide does not match any of the sequence characteristics of DNAs usually adopting the A-conformation in the crystalline state (e.g., octamers with short alternating stretches of purines and pyrimidines). In DNA-RNA chimeric duplexes, it is therefore possible that a single RNA residue can drive the conformational equilibrium toward the A-conformation.

  3. HIV biological variability unveiled: frequent isolations and chimeric receptors reveal unprecedented variation of coreceptor use.

    PubMed

    Karlsson, Ingrid; Antonsson, Liselotte; Shi, Yu; Karlsson, Anders; Albert, Jan; Leitner, Thomas; Olde, Björn; Owman, Christer; Fenyö, Eva Maria

    2003-12-05

    To follow the evolution of coreceptor use in HIV-1-infected individuals with varying rates of disease progression. The coreceptor use of 278 sequential HIV-1 isolates from 23 individuals was tested by infection of two human cell lines, U87.CD4 and GHOST(3), expressing CD4 and CCR1, CCR2b, CCR3, CCR5, CXCR4, CXCR6 or BOB. Differences in coreceptor use were further dissected by testing chimeric coreceptors constructed by exchanging successively larger parts of CCR5 for corresponding regions of CXCR4. Three patterns of coreceptor use were distinguished: no evolution, evolution to CXCR4 use, and fluctuation. No evolution with stable CCR5 use (R5 phenotype) was linked to slow progression over 8-10 years in four patients. CXCR4-using virus was present from the onset (five patients) or appeared during clinical progression in all other patients, while taking zidovudine or didanosine monotherapy. The fluctuating pattern of coreceptor use, defined as reappearance of virus with R5 phenotype, was observed in five patients and, interestingly, followed initiation of highly active antiretroviral therapy in three of these. Monotropic R5 or X4 viruses were more selective in chimeric receptor use than R5X4 or multitropic viruses. Most importantly, the efficiency of chimeric receptor use increased over time. The increase in efficiency of chimeric receptor use allows a new interpretation of evolution of HIV-1 coreceptor use. Evolution could be a continuous process that may lead to changes in the way a coreceptor is used, with the potential of profound alteration in signalling at that receptor.

  4. Application of chimeric mice with humanized liver for study of human-specific drug metabolism.

    PubMed

    Bateman, Thomas J; Reddy, Vijay G B; Kakuni, Masakazu; Morikawa, Yoshio; Kumar, Sanjeev

    2014-06-01

    Human-specific or disproportionately abundant human metabolites of drug candidates that are not adequately formed and qualified in preclinical safety assessment species pose an important drug development challenge. Furthermore, the overall metabolic profile of drug candidates in humans is an important determinant of their drug-drug interaction susceptibility. These risks can be effectively assessed and/or mitigated if human metabolic profile of the drug candidate could reliably be determined in early development. However, currently available in vitro human models (e.g., liver microsomes, hepatocytes) are often inadequate in this regard. Furthermore, the conduct of definitive radiolabeled human ADME studies is an expensive and time-consuming endeavor that is more suited for later in development when the risk of failure has been reduced. We evaluated a recently developed chimeric mouse model with humanized liver on uPA/SCID background for its ability to predict human disposition of four model drugs (lamotrigine, diclofenac, MRK-A, and propafenone) that are known to exhibit human-specific metabolism. The results from these studies demonstrate that chimeric mice were able to reproduce the human-specific metabolite profile for lamotrigine, diclofenac, and MRK-A. In the case of propafenone, however, the human-specific metabolism was not detected as a predominant pathway, and the metabolite profiles in native and humanized mice were similar; this was attributed to the presence of residual highly active propafenone-metabolizing mouse enzymes in chimeric mice. Overall, the data indicate that the chimeric mice with humanized liver have the potential to be a useful tool for the prediction of human-specific metabolism of xenobiotics and warrant further investigation.

  5. Mouse x pig chimeric antibodies expressed in Baculovirus retain the same properties of their parent antibodies.

    PubMed

    Jar, Ana M; Osorio, Fernando A; López, Osvaldo J

    2009-01-01

    The development of hybridoma and recombinant DNA technologies has made it possible to use antibodies against cancer, autoimmune disorders, and infectious diseases in humans. These advances in therapy, as well as immunoprophylaxis, could also make it possible to use these technologies in agricultural species of economic importance such as pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus causing very important economic losses to the industry. Passive transfer of antibodies obtained by biotechnology could be used in the future to complement or replace vaccination against this and other pig pathogens. To this end, we constructed and studied the properties of chimeric mouse x pig anti-PRRSV antibodies. We cloned the constant regions of gamma-1 and gamma-2 heavy chains and the lambda light chain of pig antibodies in frame with the variable regions of heavy and light chains of mouse monoclonal antibody ISU25C1, which has neutralizing activity against PRRSV. The coding regions for chimeric IgG1 and IgG2 were expressed in a baculovirus expression system. Both chimeric antibodies recognized PRRSV in ELISA as well as in a Western-blot format and, more importantly, were able to neutralize PRRSV in the same fashion as the parent mouse monoclonal antibody ISU25C1. In addition, we show that both pig IgG1 and IgG2 antibodies could bind complement component C1q, with IgG2 being more efficient than IgG1 in binding C1q. Expressing chimeric pig antibodies with protective capabilities offers a new alternative strategy for infectious disease control in domestic pigs.

  6. A chimeric human-mouse model of Sjögren's syndrome.

    PubMed

    Young, Nicholas A; Wu, Lai-Chu; Bruss, Michael; Kaffenberger, Benjamin H; Hampton, Jeffrey; Bolon, Brad; Jarjour, Wael N

    2015-01-01

    Despite recent advances in the understanding of Sjögren's Syndrome (SjS), the pathogenic mechanisms remain elusive and an ideal model for early drug discovery is not yet available. To establish a humanized mouse model of SjS, peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients with SjS were transferred into immunodeficient NOD-scid IL-2rγ(null) mouse recipients to produce chimeric mice. While no difference was observed in the distribution of cells, chimeric mice transferred with PBMCs from SjS patients produced enhanced cytokine levels, most significantly IFN-γ and IL-10. Histological examination revealed enhanced inflammatory responses in the lacrimal and salivary glands of SjS chimeras, as measured by digital image analysis and blinded histopathological scoring. Infiltrates were primarily CD4+, with minimal detection of CD8+ T-cells and B-cells. These results demonstrate a novel chimeric mouse model of human SjS that provides a unique in vivo environment to test experimental therapeutics and investigate T-cell disease pathology.

  7. Venturing in coral larval chimerism: a compact functional domain with fostered genotypic diversity

    NASA Astrophysics Data System (ADS)

    Rinkevich, Baruch; Shaish, Lee; Douek, Jacob; Ben-Shlomo, Rachel

    2016-01-01

    The globally distributed coral species Pocillopora damicornis is known to release either sexual or asexual derived planula-larvae in various reef locations. Using microsatellite loci as markers, we documented the release of asexually derived chimeric larvae (CL), originating from mosaicked maternal colonies that were also chimeras, at Thai and Philippines reefs. The CL, each presenting different combinations of maternal genotypic constituents, create genetically-complex sets of asexual propagules. This novel mode of inheritance in corals challenges classical postulations of sexual/asexual reproduction traits, as asexual derived CL represent an alliance between genotypes that significantly sways the recruits’ absolute fitness. This type of inherited chimerism, while enhancing intra-entity genetic heterogeneity, is an evolutionary tactic used to increase genetic-heterogeneity, primarily in new areas colonized by a limited number of larvae. Chimerism may also facilitate combat global change impacts by exhibiting adjustable genomic combinations of within-chimera traits that could withstand alterable environmental pressures, helping Pocillopora become a successful cosmopolitan species.

  8. Mast Cell Targeted Chimeric Toxin Can Be Developed as an Adjunctive Therapy in Colon Cancer Treatment

    PubMed Central

    Wang, Shan; Li, Linmei; Shi, Renren; Liu, Xueting; Zhang, Junyan; Zou, Zehong; Hao, Zhuofang; Tao, Ailin

    2016-01-01

    The association of colitis with colorectal cancer has become increasingly clear with mast cells being identified as important inflammatory cells in the process. In view of the relationship between mast cells and cancer, we studied the effect and mechanisms of mast cells in the development of colon cancer. Functional and mechanistic insights were gained from ex vivo and in vivo studies of cell interactions between mast cells and CT26 cells. Further evidence was reversely obtained in studies of mast cell targeted Fcε-PE40 chimeric toxin. Experiments revealed mast cells could induce colon tumor cell proliferation and invasion. Cancer progression was found to be related to the density of mast cells in colonic submucosa. The activation of MAPK, Rho-GTPase, and STAT pathways in colon cancer cells was triggered by mast cells during cell-to-cell interaction. Lastly, using an Fcε-PE40 chimeric toxin we constructed, we confirmed the promoting effect of mast cells in development of colon cancer. Mast cells are a promoting factor of colon cancer and thus also a potential therapeutic target. The Fcε-PE40 chimeric toxin targeting mast cells could effectively prevent colon cancer in vitro and in vivo. Consequently, these data may demonstrate a novel immunotherapeutic approach for the treatment of tumors. PMID:26978404

  9. Development of a high-throughput microfluidic integrated microarray for the detection of chimeric bioweapons.

    SciTech Connect

    Sheppod, Timothy; Satterfield, Brent; Hukari, Kyle W.; West, Jason A. A.; Hux, Gary A.

    2006-10-01

    The advancement of DNA cloning has significantly augmented the potential threat of a focused bioweapon assault, such as a terrorist attack. With current DNA cloning techniques, toxin genes from the most dangerous (but environmentally labile) bacterial or viral organism can now be selected and inserted into robust organism to produce an infinite number of deadly chimeric bioweapons. In order to neutralize such a threat, accurate detection of the expressed toxin genes, rather than classification on strain or genealogical decent of these organisms, is critical. The development of a high-throughput microarray approach will enable the detection of unknowns chimeric bioweapons. The development of a high-throughput microarray approach will enable the detection of unknown bioweapons. We have developed a unique microfluidic approach to capture and concentrate these threat genes (mRNA's) upto a 30 fold concentration. These captured oligonucleotides can then be used to synthesize in situ oligonucleotide copies (cDNA probes) of the captured genes. An integrated microfluidic architecture will enable us to control flows of reagents, perform clean-up steps and finally elute nanoliter volumes of synthesized oligonucleotides probes. The integrated approach has enabled a process where chimeric or conventional bioweapons can rapidly be identified based on their toxic function, rather than being restricted to information that may not identify the critical nature of the threat.

  10. Functional rescue of dystrophin-deficient mdx mice by a chimeric peptide-PMO.

    PubMed

    Yin, Haifang; Moulton, Hong M; Betts, Corinne; Merritt, Thomas; Seow, Yiqi; Ashraf, Shirin; Wang, Qingsong; Boutilier, Jordan; Wood, Matthew Ja

    2010-10-01

    Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy.

  11. Functional Rescue of Dystrophin-deficient mdx Mice by a Chimeric Peptide-PMO

    PubMed Central

    Yin, HaiFang; Moulton, Hong M; Betts, Corinne; Merritt, Thomas; Seow, Yiqi; Ashraf, Shirin; Wang, QingSong; Boutilier, Jordan; Wood, Matthew JA

    2010-01-01

    Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy. PMID:20700113

  12. Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases*

    PubMed Central

    Klaus, Maja; Ostrowski, Matthew P.; Austerjost, Jonas; Robbins, Thomas; Lowry, Brian; Cane, David E.; Khosla, Chaitan

    2016-01-01

    The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs. PMID:27246853

  13. Assembling Single-Cell Genomes and Mini-Metagenomes From Chimeric MDA Products

    PubMed Central

    Nurk, Sergey; Bankevich, Anton; Antipov, Dmitry; Gurevich, Alexey A.; Korobeynikov, Anton; Lapidus, Alla; Prjibelski, Andrey D.; Pyshkin, Alexey; Sirotkin, Alexander; Sirotkin, Yakov; Stepanauskas, Ramunas; Clingenpeel, Scott R.; Woyke, Tanja; Mclean, Jeffrey S.; Lasken, Roger; Alekseyev, Max A.; Pevzner, Pavel A.

    2013-01-01

    Abstract Recent advances in single-cell genomics provide an alternative to largely gene-centric metagenomics studies, enabling whole-genome sequencing of uncultivated bacteria. However, single-cell assembly projects are challenging due to (i) the highly nonuniform read coverage and (ii) a greatly elevated number of chimeric reads and read pairs. While recently developed single-cell assemblers have addressed the former challenge, methods for assembling highly chimeric reads remain poorly explored. We present algorithms for identifying chimeric edges and resolving complex bulges in de Bruijn graphs, which significantly improve single-cell assemblies. We further describe applications of the single-cell assembler SPAdes to a new approach for capturing and sequencing “microbial dark matter” that forms small pools of randomly selected single cells (called a mini-metagenome) and further sequences all genomes from the mini-metagenome at once. On single-cell bacterial datasets, SPAdes improves on the recently developed E+V-SC and IDBA-UD assemblers specifically designed for single-cell sequencing. For standard (cultivated monostrain) datasets, SPAdes also improves on A5, ABySS, CLC, EULER-SR, Ray, SOAPdenovo, and Velvet. Thus, recently developed single-cell assemblers not only enable single-cell sequencing, but also improve on conventional assemblers on their own turf. SPAdes is available for free online download under a GPLv2 license. PMID:24093227

  14. Hotspot Selective Preference of the Chimeric Sequences Formed in Multiple Displacement Amplification

    PubMed Central

    Tu, Jing; Lu, Na; Duan, Mengqin; Huang, Mengting; Chen, Liang; Li, Junji; Guo, Jing; Lu, Zuhong

    2017-01-01

    Multiple displacement amplification (MDA) is considered to be a conventional approach to comprehensive amplification from low input DNA. The chimeric reads generated in MDA lead to severe disruption in some studies, including those focusing on heterogeneity, structural variation, and genetic recombination. Meanwhile, the generation of by-products gives a new approach to gain insights into the reaction process of φ29 polymerase. Here, we analyzed 36.7 million chimeras and screened 196 billion chimeric hotspots in the human genome, as well as evaluating the hotspot selective preference of chimeras. No significant preference was captured in the distributions of chimeras and hotspots among chromosomes. Hotspots with overlaps for 12–13 nucleotides (nt) were most likely to be selected as templates in chimera generation. Meanwhile, a regularly selective preference was noticed in overlap GC content. The preferences in overlap length and GC content was shown to be pertinent to the sequence denaturation temperature, which pointed out the optimization direction for reducing chimeras. Distance preference between two segments of chimeras was 80–280 nt. The analysis is beneficial for reducing the chimeras in MDA, and the characterization of MDA chimeras is helpful in distinguishing MDA chimeras from chimeric sequences caused by disease. PMID:28245591

  15. Expression and secretion of aequorin as a chimeric antibody by means of a mammalian expression vector.

    PubMed Central

    Casadei, J; Powell, M J; Kenten, J H

    1990-01-01

    A fusion protein has been expressed from the relevant genes in mammalian cells consisting of the photoprotein aequorin and an anti-4-hydroxy-3-nitrophenacetyl antibody gene. This chimeric antibody has allowed the development of a sensitive luminescent immunoassay. Initially the cDNA of the photoprotein aequorin from Aequorea victoria was cloned and expressed in Escherichia coli. The gene was expressed as apoaequorin and, by using luciferin isolated from Renilla reniformis, its activity was found essentially identical to native aequorin. The aequorin gene was subcloned into a mammalian expression vector to produce a fusion protein directing secretion of apoaequorin; the aequorin gene was fused to the 3' terminus of an immunoglobulin heavy-chain gene that directed expression of an anti-4-hydroxy-3-nitrophenacetyl antibody. The gene fusion contained the variable region, the constant region domain 1, and part of domain 2 for the IgG2b mouse immunoglobulin, followed by the aequorin gene. Transfection of the chimeric gene into a cell line expressing the complementary lambda 1 light chain, J558L, allowed recovery of a chimeric antibody with binding specificity for the 4-hydroxy-3-nitrophenacetyl group and the related 4-hydroxy-3-iodo-5-nitrophenacetyl hapten. The Ca2(+)-dependent bioluminescent activity of aequorin was also recovered. Images PMID:2315301

  16. Venturing in coral larval chimerism: a compact functional domain with fostered genotypic diversity

    PubMed Central

    Rinkevich, Baruch; Shaish, Lee; Douek, Jacob; Ben-Shlomo, Rachel

    2016-01-01

    The globally distributed coral species Pocillopora damicornis is known to release either sexual or asexual derived planula-larvae in various reef locations. Using microsatellite loci as markers, we documented the release of asexually derived chimeric larvae (CL), originating from mosaicked maternal colonies that were also chimeras, at Thai and Philippines reefs. The CL, each presenting different combinations of maternal genotypic constituents, create genetically-complex sets of asexual propagules. This novel mode of inheritance in corals challenges classical postulations of sexual/asexual reproduction traits, as asexual derived CL represent an alliance between genotypes that significantly sways the recruits’ absolute fitness. This type of inherited chimerism, while enhancing intra-entity genetic heterogeneity, is an evolutionary tactic used to increase genetic-heterogeneity, primarily in new areas colonized by a limited number of larvae. Chimerism may also facilitate combat global change impacts by exhibiting adjustable genomic combinations of within-chimera traits that could withstand alterable environmental pressures, helping Pocillopora become a successful cosmopolitan species. PMID:26758405

  17. Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreceptors.

    PubMed

    Wang, Enxiu; Wang, Liang-Chuan; Tsai, Ching-Yi; Bhoj, Vijay; Gershenson, Zack; Moon, Edmund; Newick, Kheng; Sun, Jing; Lo, Albert; Baradet, Timothy; Feldman, Michael D; Barrett, David; Puré, Ellen; Albelda, Steven; Milone, Michael C

    2015-07-01

    Chimeric antigen receptors (CAR) bearing an antigen-binding domain linked in cis to the cytoplasmic domains of CD3ζ and costimulatory receptors have provided a potent method for engineering T-cell cytotoxicity toward B-cell leukemia and lymphoma. However, resistance to immunotherapy due to loss of T-cell effector function remains a significant barrier, especially in solid malignancies. We describe an alternative chimeric immunoreceptor design in which we have fused a single-chain variable fragment for antigen recognition to the transmembrane and cytoplasmic domains of KIR2DS2, a stimulatory killer immunoglobulin-like receptor (KIR). We show that this simple, KIR-based CAR (KIR-CAR) triggers robust antigen-specific proliferation and effector function in vitro when introduced into human T cells with DAP12, an immunotyrosine-based activation motifs-containing adaptor. T cells modified to express a KIR-CAR and DAP12 exhibit superior antitumor activity compared with standard first- and second-generation CD3ζ-based CARs in a xenograft model of mesothelioma highly resistant to immunotherapy. The enhanced antitumor activity is associated with improved retention of chimeric immunoreceptor expression and improved effector function of isolated tumor-infiltrating lymphocytes. These results support the exploration of KIR-CARs for adoptive T-cell immunotherapy, particularly in immunotherapy-resistant solid tumors.

  18. Immunotherapy for cancer: construction, expression and functional characterization of chimeric antibodies.

    PubMed

    Motmans, K; Thirion, S; Heyligen, H; Janssens, J; Raus, J; Vandevyver, C

    1996-12-01

    Monoclonal antibodies (Mabs) are a potential key component for the treatment of cancer, because of their specificity and multiple effector functions. Hybridoma technology and progress in genetic engineering made it possible to customize antibody molecules, rendering them more suitable for selective application. A widely used technique is the construction of mouse-human hybrid molecules by recombinant DNA techniques. These so-called chimeric antibodies contain the murine variable (V) regions fused to the human constant (C) regions. In this report, a general approach is described for the production of chimeric antibodies. The gene segments encoding the murine variable heavy and light chain are isolated by the polymerase chain reaction and cloned into expression vectors containing the human gamma 1 heavy chain gene and the human K light chain gene, respectively. Subsequently, these constructs are transfected into a non-Ig-producing murine hybridoma, eg SP2/0 cells. The in vitro study of the functional characteristics and biological properties of the thus obtained chimeric antibodies are discussed.

  19. Human-animal chimeras: ethical issues about farming chimeric animals bearing human organs.

    PubMed

    Bourret, Rodolphe; Martinez, Eric; Vialla, François; Giquel, Chloé; Thonnat-Marin, Aurélie; De Vos, John

    2016-06-29

    Recent advances in stem cells and gene engineering have paved the way for the generation of interspecies chimeras, such as animals bearing an organ from another species. The production of a rat pancreas by a mouse has demonstrated the feasibility of this approach. The next step will be the generation of larger chimeric animals, such as pigs bearing human organs. Because of the dramatic organ shortage for transplantation, the medical needs for such a transgressive practice are indisputable. However, there are serious technical barriers and complex ethical issues that must be discussed and solved before producing human organs in animals. The main ethical issues are the risks of consciousness and of human features in the chimeric animal due to a too high contribution of human cells to the brain, in the first case, or for instance to limbs, in the second. Another critical point concerns the production of human gametes by such chimeric animals. These worst-case scenarios are obviously unacceptable and must be strictly monitored by careful risk assessment, and, if necessary, technically prevented. The public must be associated with this ethical debate. Scientists and physicians have a critical role in explaining the medical needs, the advantages and limits of this potential medical procedure, and the ethical boundaries that must not be trespassed. If these prerequisites are met, acceptance of such a new, borderline medical procedure may prevail, as happened before for in-vitro fertilization or preimplantation genetic diagnosis.

  20. [Blood cell chimerism in dizygotic twins conceived by in vitro fertilization].

    PubMed

    Martos-Moreno, G Á; Campos, C; Flores, R; Yturriaga, R; Pérez-Jurado, L A; Argente, J

    2013-10-01

    We present a case of hematopoietic chimerism in dizygotic twins (male and female) conceived by in vitro fertilization (IVF). At 8 years of age a blood karyotype was performed on the female due to the presence of clitoromegaly. Two different lines: 46,XX (53%) and 46,XY (47%) were found. FISH studies confirmed the presence of the SRY gene in 46,XY cells. Karyotyping of the male showed two different lines: 46,XY (58%) and 46,XX (42%). SRY gene was present in 46,XY cells. Microsatellite analyses of blood DNA revealed tetra-allelic contribution at some autosomal loci with similar proportions of maternal and paternal alleles and X/Y chromosome dose. FISH in buccal mucous showed that all cells from the female were 46,XX and those from the male 46,XY. The gonadal karyotype in the female was 46,XX without SRY. Hence, we report 46,XX/46,XY chimerism in dizygotic twins. Blood chimerism was confirmed by performing FISH on the buccal cells of the patients.

  1. A chimeric NST repressor has the potential to improve glucose productivity from plant cell walls.

    PubMed

    Iwase, Akira; Hideno, Akihiro; Watanabe, Keiji; Mitsuda, Nobutaka; Ohme-Takagi, Masaru

    2009-07-15

    Bioethanol might be produced more economically and with less ecological impact (with reduced exploitation of food crops) if we could increase the production of glucose from the cellulosic materials in plant cell walls. However, plant cell walls are relatively resistant to enzymatic and physicochemical hydrolysis and, therefore, it is necessary to develop methods for reducing such resistance. Changes in plant cell wall materials, by genetic engineering, that render them more easily hydrolyzable to glucose might be a valuable approach to this problem. We showed previously that, in Arabidopsis, NAC secondary wall thickening-promoting factor1 (NST1) and NST3 are key regulators of secondary wall formation. We report here that transgenic Arabidopsis plants that expressed a chimeric repressor derived from NST1 produced cell wall materials that were twice as susceptible to both enzymatic and physicochemical hydrolysis as those from wild-type plants. The yields of glucose from both fresh and dry biomass were increased in the chimeric repressor lines. Use of the NST1 chimeric repressor might enhance production of glucose from plant cell walls, by changing the nature of the cell walls themselves.

  2. Construction of a photo-responsive chimeric histidine kinase in Escherichia coli.

    PubMed

    Hori, Mayuko; Oka, Shyunsuke; Sugie, Yoshimi; Ohtsuka, Hokuto; Aiba, Hirofumi

    2017-01-31

    Two-component signal transduction systems (TCS), that are also referred to as His to Asp phosphorelay systems, are involved in widespread cellular responses to diverse signals from bacteria to plants. Previously, we succeeded in reconstructing a cyanobacterial photo-perception system in Escherichia coli by employing a CcaS-CcaR two-component system from Nostoc punctiforme. In this study, we have added a photo-responsive ability to ArcB-ArcA (anoxic redox control) TCS of E. coli by fusing a cyanobacterial photoreceptor domain of CcaS with an intracellular histidine kinase (HK) domain of ArcB. For this, we constructed several chimeric HKs between CcaS and ArcB and found that one chimeric HK, named ArcaS9, has a photo-responsive ability. When ArcaS9 was expressed with an ArcA response regulator in E. coli expressing phycocyanobilin (PCB)-producing enzymes, the expression of sdh, a target gene of ArcB-ArcA TCS was regulated in a light-color-dependent manner. Thus we succeeded in endowing E. coli HK with a photo-responsive ability. This provides an insight into how the sensing ability of HK can be manipulated by a chimeric construct.

  3. Characterization of a chimeric monoclonal antibody against the insulin-like growth factor-I receptor.

    PubMed

    Zhang, Mei-Yun; Feng, Yang; Wang, Yanping; Dimitrov, Dimiter S

    2009-01-01

    The insulin-like growth factors (IGFs) signaling system has been shown to play important roles in neoplasia. The IGF receptor type 1 (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and there is substantial experimental and clinical evidence that targeting IGF-IR is a promising therapeutic strategy against cancer. It has been previously reported that a mouse monoclonal antibody (mAb), 4G11, blocked IGF-I binding to IGF-IR and downregulated the IGF-IR in MCF-7 cells. We cloned this antibody, constructed a human-mouse chimeric antibody, designated m590, and characterized it. The chimeric IgG1 m590 bound to cell-associated IGF-IR on NWT c43 stably transfected cells and MCF-7 breast cancer cells as efficiently as the parental murine antibody. Using purified IGF-IR extracellular domains, we found that both the chimeric m590 and the parental 4G11 antibodies bind to conformational epitopes on IGF-IR. Neither of these antibodies bound to the insulin receptor (IR) ectodomain. Furthermore, IgG1 m590 blocked the binding of IGF-I and IGF-II to IGF-IR, and inhibited both IGF-I and IGF-II induced phosphorylation of IGF-IR in MCF-7 cells. These results suggest that m590 could be an useful antibody in diagnosis and treatment of cancer, as well as a research tool.

  4. Characterization of a chimeric monoclonal antibody against the insulin-like growth factor-I receptor

    PubMed Central

    Feng, Yang; Wang, Yanping; Dimitrov, Dimiter S

    2009-01-01

    The insulin-like growth factors (IGFs) signaling system has been shown to play important roles in neoplasia. The IGF receptor type 1 (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and there is substantial experimental and clinical evidence that targeting IGF-IR is a promising therapeutic strategy against cancer. It has been previously reported that a mouse monoclonal antibody (mAb), 4G11, blocked IGF-I binding to IGF-IR and downregulated the IGF-IR in MCF-7 cells. We cloned this antibody, constructed a human-mouse chimeric antibody, designated m590, and characterized it. The chimeric IgG1 m590 bound to cell-associated IGF-IR on NWT c43 stably transfected cells and MCF-7 breast cancer cells as efficiently as the parental murine antibody. Using purified IGF-IR extracellular domains, we found that both the chimeric m590 and the parental 4G11 antibodies bind to conformational epitopes on IGF-IR. Neither of these antibodies bound to the insulin receptor (IR) ectodomain. Furthermore, IgG1 m590 blocked the binding of IGF-I and IGF-II to IGF-IR, and inhibited both IGF-I and IGF-II induced phosphorylation of IGF-IR in MCF-7 cells. These results suggest that m590 could be an useful antibody in diagnosis and treatment of cancer, as well as a research tool. PMID:20065647

  5. Characterization of Moloney murine leukaemia virus/avian myeloblastosis virus chimeric reverse transcriptases.

    PubMed

    Yasukawa, Kiyoshi; Mizuno, Masaki; Inouye, Kuniyo

    2009-03-01

    Reverse transcriptases (RTs) from Moloney murine leukaemia virus (MMLV) and avian myeloblastosis virus (AMV) contain all the fingers, palm, thumb, connection and RNase H domains. The fingers, palm and thumb domains are thought to be involved in the reverse transcription activity, and the RNase H domain is in the RNase H activity. In this study, we characterized four chimeric RTs which comprise one of the fingers, palm, thumb and RNase H domains originated from AMV RT and the other three and connection domains originated from MMLV RT. Unexpectedly, all chimeric RTs exhibited the same characteristics: their specific reverse transcription activities decreased to less than 0.1% of that of MMLV RT, while their specific RNase H activities were approximately 20% of that of MMLV RT. The decreases in the two activities of the chimeric RTs were ascribed to the decreases in k(cat). Based on that the reverse transcription activity of MMLV RT was impaired by substituting its RNase H domain with that from AMV RT, we propose that in MMLV RT, there might be an interaction between the fingers/palm/thumb domain and the RNase H domain.

  6. Japanese encephalitis virus vaccine candidates generated by chimerization with dengue virus type 4.

    PubMed

    Gromowski, Gregory D; Firestone, Cai-Yen; Hanson, Christopher T; Whitehead, Stephen S

    2014-05-23

    Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis worldwide and vaccination is one of the most effective ways to prevent disease. A suitable live-attenuated JEV vaccine could be formulated with a live-attenuated tetravalent dengue vaccine for the control of these viruses in endemic areas. Toward this goal, we generated chimeric virus vaccine candidates by replacing the precursor membrane (prM) and envelope (E) protein structural genes of recombinant dengue virus type 4 (rDEN4) or attenuated vaccine candidate rDEN4Δ30 with those of wild-type JEV strain India/78. Mutations were engineered in E, NS3 and NS4B protein genes to improve replication in Vero cells. The chimeric viruses were attenuated in mice and some elicited modest but protective levels of immunity after a single dose. One particular chimeric virus, bearing E protein mutation Q264H, replicated to higher titer in tissue culture and was significantly more immunogenic in mice. The results are compared with live-attenuated JEV vaccine strain SA14-14-2. Published by Elsevier Ltd.

  7. Zygotes segregate entire parental genomes in distinct blastomere lineages causing cleavage-stage chimerism and mixoploidy

    PubMed Central

    Destouni, Aspasia; Zamani Esteki, Masoud; Catteeuw, Maaike; Tšuiko, Olga; Dimitriadou, Eftychia; Smits, Katrien; Kurg, Ants; Salumets, Andres; Van Soom, Ann; Voet, Thierry; Vermeesch, Joris R.

    2016-01-01

    Dramatic genome dynamics, such as chromosome instability, contribute to the remarkable genomic heterogeneity among the blastomeres comprising a single embryo during human preimplantation development. This heterogeneity, when compatible with life, manifests as constitutional mosaicism, chimerism, and mixoploidy in live-born individuals. Chimerism and mixoploidy are defined by the presence of cell lineages with different parental genomes or different ploidy states in a single individual, respectively. Our knowledge of their mechanistic origin results from indirect observations, often when the cell lineages have been subject to rigorous selective pressure during development. Here, we applied haplarithmisis to infer the haplotypes and the copy number of parental genomes in 116 single blastomeres comprising entire preimplantation bovine embryos (n = 23) following in vitro fertilization. We not only demonstrate that chromosome instability is conserved between bovine and human cleavage embryos, but we also discovered that zygotes can spontaneously segregate entire parental genomes into different cell lineages during the first post-zygotic cleavage division. Parental genome segregation was not exclusively triggered by abnormal fertilizations leading to triploid zygotes, but also normally fertilized zygotes can spontaneously segregate entire parental genomes into different cell lineages during cleavage of the zygote. We coin the term “heterogoneic division” to indicate the events leading to noncanonical zygotic cytokinesis, segregating the parental genomes into distinct cell lineages. Persistence of those cell lines during development is a likely cause of chimerism and mixoploidy in mammals. PMID:27197242

  8. Overexpression of a Chimeric Gene, OsDST-SRDX, Improved Salt Tolerance of Perennial Ryegrass

    PubMed Central

    Cen, Huifang; Ye, Wenxing; Liu, Yanrong; Li, Dayong; Wang, Kexin; Zhang, Wanjun

    2016-01-01

    The Drought and Salt Tolerance gene (DST) encodes a C2H2 zinc finger transcription factor, which negatively regulates salt tolerance in rice (Oryza sativa). Phylogenetic analysis of six homologues of DST genes in different plant species revealed that DST genes were conserved evolutionarily. Here, the rice DST gene was linked to an SRDX domain for gene expression repression based on the Chimeric REpressor gene-Silencing Technology (CRES-T) to make a chimeric gene (OsDST-SRDX) construct and introduced into perennial ryegrass by Agrobacterium-mediated transformation. Integration and expression of the OsDST-SRDX in transgenic plants were tested by PCR and RT-PCR, respectively. Transgenic lines overexpressing the OsDST-SRDX fusion gene showed obvious phenotypic differences and clear resistance to salt-shock and to continuous salt stresses compared to non-transgenic plants. Physiological analyses including relative leaf water content, electrolyte leakage, proline content, malondialdehyde (MDA) content, H2O2 content and sodium and potassium accumulation indicated that the OsDST-SRDX fusion gene enhanced salt tolerance in transgenic perennial ryegrass by altering a wide range of physiological responses. To our best knowledge this study is the first report of utilizing Chimeric Repressor gene-Silencing Technology (CRES-T) in turfgrass and forage species for salt-tolerance improvement. PMID:27251327

  9. Chimeric Aptamer-Gelatin Hydrogels as an Extracellular Matrix Mimic for Loading Cells and Growth Factors

    PubMed Central

    Zhang, Xiaolong; Battig, Mark R.; Chen, Niancao; Gaddes, Erin R.; Duncan, Katelyn L.; Wang, Yong

    2016-01-01

    It is important to synthesize materials to recapitulate critical functions of biological systems for a variety of applications such as tissue engineering and regenerative medicine. The purpose of this study was to synthesize a chimeric hydrogel as a promising extracellular matrix (ECM) mimic using gelatin, a nucleic acid aptamer and polyethylene glycol (PEG). This hydrogel had a macroporous structure that was highly permeable for fast molecular transport. Despite its high permeability, it could strongly sequester and sustainably release growth factors with high bioactivity. Notably, growth factors retained in the hydrogel could maintain ~50% bioactivity during a 14-day release test. It also provided cells with effective binding sites, which led to high efficiency of cell loading into the macroporous hydrogel matrix. When cells and growth factors were co-loaded into the chimeric hydrogel, living cells could still be observed by day 14 in a static serum-reduced culture condition. Thus, this chimeric aptamer-gelatin hydrogel constitutes a promising biomolecular ECM mimic for loading cells and growth factors. PMID:26791559

  10. Confined blood chimerism in a monochorionic dizygotic sex discordant twin pregnancy conceived after induced ovulation.

    PubMed

    Mayeur Le Bras, Anne; Petit, François; Benachi, Alexandra; Bedel, Bettina; Oucherif, Salima; Martinovic, Jelena; Armanet, Narjes; Tosca, Lucie; Gautier, Valérie; Parisot, Frédéric; Labrune, Philippe; Tachdjian, Gérard; Brisset, Sophie

    2016-04-01

    Monochorionic twins are generally considered as a monozygotic twin pregnancy. However, several cases of monochorial dizygotic twin pregnancies have been reported. We report on a rare case of monochorionic dizygotic twin pregnancy conceived after induced ovulation in a 32-year-old woman. The diagnosis was made on morphological ultrasound examination at 18+4 weeks of gestation, showing two fetuses with discordant sex. The amniocentesis was declined by the patient. The monochorionic status was confirmed after a histopathalogical study of the placenta. At delivery, both a phenotypically normal boy and a phenotypically normal girl without sexual abnormality were observed. This analysis also revealed the presence of vascular anastomoses between both fetal circulations. Postnatal cytogenetic analyses indicated the presence of a chimerism in peripheral blood lymphocytes. This chimerism was not observed in cells obtained from a buccal swab. Molecular determination of zygosity confirmed the existence of the confined peripheral blood chimerism with the presence of four parental alleles. We report on a case of monochorionic dizygotic twin pregnancy. This observation underlies the need to carefully assess twin pregnancies, especially when obtained after assisted reproductive technology. © 2016 Wiley Periodicals, Inc.

  11. Multiple Layers of Chimerism in a Single-Stranded DNA Virus Discovered by Deep Sequencing

    PubMed Central

    Krupovic, Mart; Zhi, Ning; Li, Jungang; Hu, Gangqing; Koonin, Eugene V.; Wong, Susan; Shevchenko, Sofiya; Zhao, Keji; Young, Neal S.

    2015-01-01

    Viruses with single-stranded (ss) DNA genomes infect hosts in all three domains of life and include many medically, ecologically, and economically important pathogens. Recently, a new group of ssDNA viruses with chimeric genomes has been discovered through viral metagenomics. These chimeric viruses combine capsid protein genes and replicative protein genes that, respectively, appear to have been inherited from viruses with positive-strand RNA genomes, such as tombusviruses, and ssDNA genomes, such as circoviruses, nanoviruses or geminiviruses. Here, we describe the genome sequence of a new representative of this virus group and reveal an additional layer of chimerism among ssDNA viruses. We show that not only do these viruses encompass genes for capsid proteins and replicative proteins that have distinct evolutionary histories, but also the replicative genes themselves are chimeras of functional domains inherited from viruses of different families. Our results underscore the importance of horizontal gene transfer in the evolution of ssDNA viruses and the role of genetic recombination in the emergence of novel virus groups. PMID:25840414

  12. Genetic repair of mutations in plant cell-free extracts directed by specific chimeric oligonucleotides.

    PubMed

    Rice, M C; May, G D; Kipp, P B; Parekh, H; Kmiec, E B

    2000-06-01

    Chimeric oligonucleotides are synthetic molecules comprised of RNA and DNA bases assembled in a double hairpin conformation. These molecules have been shown to direct gene conversion events in mammalian cells and animals through a process involving at least one protein from the DNA mismatch repair pathway. The mechanism of action for gene repair in mammalian cells has been partially elucidated through the use of a cell-free extract system. Recent experiments have expanded the utility of chimeric oligonucleotides to plants and have demonstrated genotypic and phenotypic conversion, as well as Mendelian transmission. Although these experiments showed correction of point and frameshift mutations, the biochemical and mechanistic aspects of the process were not addressed. In this paper, we describe the establishment of cell-free extract systems from maize (Zea mays), banana (Musa acuminata cv Rasthali), and tobacco (Nicotiana tabacum). Using a genetic readout system in bacteria and chimeric oligonucleotides designed to direct the conversion of mutations in antibiotic-resistant genes, we demonstrate gene repair of point and frameshift mutations. Whereas extracts from banana and maize catalyzed repair of mutations in a precise fashion, cell-free extracts prepared from tobacco exhibited either partial repair or non-targeted nucleotide conversion. In addition, an all-DNA hairpin molecule also mediated repair albeit in an imprecise fashion in all cell-free extracts tested. This system enables the mechanistic study of gene repair in plants and may facilitate the identification of DNA repair proteins operating in plant cells.

  13. Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation

    PubMed Central

    Stahl, Tanja; Rothe, Caroline; Böhme, Manja U.; Kohl, Aloisa; Kröger, Nicolaus; Fehse, Boris

    2016-01-01

    Accurate and sensitive determination of hematopoietic chimerism is a crucial diagnostic measure after allogeneic stem cell transplantation to monitor engraftment and potentially residual disease. Short tandem repeat (STR) amplification, the current “gold standard” for chimerism assessment facilitates reliable accuracy, but is hampered by its limited sensitivity (≥1%). Digital PCR (dPCR) has been shown to combine exact quantification and high reproducibility over a very wide measurement range with excellent sensitivity (routinely ≤0.1%) and thus represents a promising alternative to STR analysis. We here aimed at developing a whole panel of digital-PCR based assays for routine diagnostic. To this end, we tested suitability of 52 deletion/insertion polymorphisms (DIPs) for duplex analysis in combination with either a reference gene or a Y-chromosome specific PCR. Twenty-nine DIPs with high power of discrimination and good performance were identified, optimized and technically validated. We tested the newly established assays on retrospective patient samples that were in parallel also measured by STR amplification and found excellent correlation. Finally, a screening plate for initial genotyping with DIP-specific duplex dPCR assays was designed for convenient assay selection. In conclusion, we have established a comprehensive dPCR system for precise and high-sensitivity measurement of hematopoietic chimerism, which should be highly useful for clinical routine diagnostics. PMID:27618030

  14. HomologMiner: looking for homologous genomic groups in whole genomes.

    PubMed

    Hou, Minmei; Berman, Piotr; Hsu, Chih-Hao; Harris, Robert S

    2007-04-15

    Complex genomes contain numerous repeated sequences, and genomic duplication is believed to be a main evolutionary mechanism to obtain new functions. Several tools are available for de novo repeat sequence identification, and many approaches exist for clustering homologous protein sequences. We present an efficient new approach to identify and cluster homologous DNA sequences with high accuracy at the level of whole genomes, excluding low-complexity repeats, tandem repeats and annotated interspersed repeats. We also determine the boundaries of each group member so that it closely represents a biological unit, e.g. a complete gene, or a partial gene coding a protein domain. We developed a program called HomologMiner to identify homologous groups applicable to genome sequences that have been properly marked for low-complexity repeats and annotated interspersed repeats. We applied it to the whole genomes of human (hg17), macaque (rheMac2) and mouse (mm8). Groups obtained include gene families (e.g. olfactory receptor gene family, zinc finger families), unannotated interspersed repeats and additional homologous groups that resulted from recent segmental duplications. Our program incorporates several new methods: a new abstract definition of consistent duplicate units, a new criterion to remove moderately frequent tandem repeats, and new algorithmic techniques. We also provide preliminary analysis of the output on the three genomes mentioned above, and show several applications including identifying boundaries of tandem gene clusters and novel interspersed repeat families. All programs and datasets are downloadable from www.bx.psu.edu/miller_lab.

  15. In silico and experimental characterization of chimeric Bacillus thermocatenulatus lipase with the complete conserved pentapeptide of Candida rugosa lipase.

    PubMed

    Hosseini, Mostafa; Karkhane, Ali Asghar; Yakhchali, Bagher; Shamsara, Mehdi; Aminzadeh, Saeed; Morshedi, Dena; Haghbeen, Kamahldin; Torktaz, Ibrahim; Karimi, Esmat; Safari, Zahra

    2013-02-01

    Lipases are one of the highest value commercial enzymes as they have broad applications in detergent, food, pharmaceutical, and dairy industries. To provide chimeric Bacillus thermocatenulatus lipase (BTL2), the completely conserved pentapeptide (¹¹²Ala-His-Ser-Gln-Gly¹¹⁶) was replaced with similar sequences (²⁰⁷Gly-Glu-Ser-Ala-Gly²¹¹) of Candida rugosa lipase (CLR) at the nucleophilic elbow region. For this purpose, three mutations including A112G, H113E, and Q115A were inserted in the conserved pentapeptide sequence of btl2 gene. Based on the crystal structures of 2W22, the best structure of opened form of the chimeric lipases were garnered using the MODELLER v9.10 software. The native and chimeric lipases were docked to a set of ligands, and a trial version of Molegro Virtual Docker (MVD) software was used to obtain the energy values. Docking results confirmed chimeric lipase to be better than the native lipase. Following the in silico study, cloning experiments were conducted and expression of native and chimeric btl2 gene in Pichia pastoris was performed. The native and chimeric lipases were purified, and the effect of these mutations on characteristics of chimeric lipase studied and then compared with those of native lipase. Chimeric lipase exhibited 1.6-fold higher activity than the native lipase at 55 °C. The highest percentage of both lipases activity was observed at 60 °C and pH of 8.0. The ion Ca²⁺ slightly inhibited the activity of both lipases, whereas the organic solvent enhanced the lipase stability of chimeric lipase as compared with the native lipase. According to the results, the presence of two glycine residues at the conserved pentapeptide region of this chimeric lipase (¹¹²Gly-Glu-Ser-Ala-Gly¹¹⁶) may increase the flexibility of the nucleophilic elbow region and affect the enzyme activity level.

  16. High frequency of donor chimerism after allogeneic transplantation of CD34+-selected peripheral blood cells.

    PubMed

    Briones, J; Urbano-Ispizua, A; Lawler, M; Rozman, C; Gardiner, N; Marín, P; Salgado, C; Féliz, P; McCann, S; Montserrat, E

    1998-05-01

    Ex vivo T cell depletion of allogeneic grafts is associated with a high (up to 80%) rate of mixed chimerism (MC) posttransplantation. The number of transplanted progenitor cells is an important factor in achieving complete donor chimerism in the T cell depletion setting. Use of granulocyte colony-stimulating factor (G-CSF) peripheral blood allografts allows the administration of large numbers of CD34+ cells. We studied the chimeric status of 13 patients who received allogeneic CD34+-selected peripheral blood progenitor cell transplants (allo-PBPCTs/CD34+) from HLA-identical sibling donors. Patients were conditioned with cyclophosphamide (120 mg/kg) and total-body irradiation (13 Gy in four fractions). Apheresis products were T cell-depleted by the immunoadsorption avidin-biotin method. The median number of CD34+ and CD3+ cells infused was 2.8x10(6)/kg (range 1.9-8.6x10(6)/kg) and 0.4x10(6)/kg (range 0.3-1x10(6)/kg), respectively. Molecular analysis of the engraftment was performed using polymerase chain reaction (PCR) amplification of highly polymorphic short tandem repeat (PCR-STR) sequences in peripheral blood samples. MC was detected in two (15%) of 13 patients. These two patients relapsed at 8 and 10 months after transplant, respectively. The remaining 11 patients showed complete donor chimerism and were in clinical remission after a maximum follow-up period of 24 months (range 6-24 months). These results were compared with those obtained in 10 patients who were treated with T cell-depleted bone marrow transplantation by means of elutriation and who received the same conditioning treatment and similar amounts of CD3+ cells (median 0.45x10(6)/kg; not significant) but a lower number of CD34+ cells (median 0.8x10(6)/kg; p = 0.001). MC was documented in six of 10 patients (60%), which was significantly higher than in the allo-PBPCT/CD34+ group (p = 0.04). We conclude that a high frequency of complete donor chimerism is achieved in patients receiving allo-PBPCT/CD34

  17. PRIMO: An Interactive Homology Modeling Pipeline.

    PubMed

    Hatherley, Rowan; Brown, David K; Glenister, Michael; Tastan Bishop, Özlem

    2016-01-01

    The development of automated servers to predict the three-dimensional structure of proteins has seen much progress over the years. These servers make calculations simpler, but largely exclude users from the process. In this study, we present the PRotein Interactive MOdeling (PRIMO) pipeline for homology modeling of protein monomers. The pipeline eases the multi-step modeling process, and reduces the workload required by the user, while still allowing engagement from the user during every step. Default parameters are given for each step, which can either be modified or supplemented with additional external input. PRIMO has been designed for users of varying levels of experience with homology modeling. The pipeline incorporates a user-friendly interface that makes it easy to alter parameters used during modeling. During each stage of the modeling process, the site provides suggestions for novice users to improve the quality of their models. PRIMO provides functionality that allows users to also model ligands and ions in complex with their protein targets. Herein, we assess the accuracy of the fully automated capabilities of the server, including a comparative analysis of the available alignment programs, as well as of the refinement levels used during modeling. The tests presented here demonstrate the reliability of the PRIMO server when producing a large number of protein models. While PRIMO does focus on user involvement in the homology modeling process, the results indicate that in the presence of suitable templates, good quality models can be produced even without user intervention. This gives an idea of the base level accuracy of PRIMO, which users can improve upon by adjusting parameters in their modeling runs. The accuracy of PRIMO's automated scripts is being continuously evaluated by the CAMEO (Continuous Automated Model EvaluatiOn) project. The PRIMO site is free for non-commercial use and can be accessed at https://primo.rubi.ru.ac.za/.

  18. PRIMO: An Interactive Homology Modeling Pipeline

    PubMed Central

    Glenister, Michael

    2016-01-01

    The development of automated servers to predict the three-dimensional structure of proteins has seen much progress over the years. These servers make calculations simpler, but largely exclude users from the process. In this study, we present the PRotein Interactive MOdeling (PRIMO) pipeline for homology modeling of protein monomers. The pipeline eases the multi-step modeling process, and reduces the workload required by the user, while still allowing engagement from the user during every step. Default parameters are given for each step, which can either be modified or supplemented with additional external input. PRIMO has been designed for users of varying levels of experience with homology modeling. The pipeline incorporates a user-friendly interface that makes it easy to alter parameters used during modeling. During each stage of the modeling process, the site provides suggestions for novice users to improve the quality of their models. PRIMO provides functionality that allows users to also model ligands and ions in complex with their protein targets. Herein, we assess the accuracy of the fully automated capabilities of the server, including a comparative analysis of the available alignment programs, as well as of the refinement levels used during modeling. The tests presented here demonstrate the reliability of the PRIMO server when producing a large number of protein models. While PRIMO does focus on user involvement in the homology modeling process, the results indicate that in the presence of suitable templates, good quality models can be produced even without user intervention. This gives an idea of the base level accuracy of PRIMO, which users can improve upon by adjusting parameters in their modeling runs. The accuracy of PRIMO’s automated scripts is being continuously evaluated by the CAMEO (Continuous Automated Model EvaluatiOn) project. The PRIMO site is free for non-commercial use and can be accessed at https://primo.rubi.ru.ac.za/. PMID:27855192

  19. Fatal attraction: cytomegalovirus-encoded chemokine homologs.

    PubMed

    Saederup, N; Mocarski, E S

    2002-01-01

    Members of the cytomegalovirus (CMV) subfamily of betaherpesviruses infecting primates and rodents encode divergent proteins with sequence characteristics and activities of chemokines, a class of small, secreted proteins that control leukocyte migration and trafficking behavior. Human CMV genes UL146 and UL147 encode proteins with sequence characteristics of CXC chemokines, whereas, murine CMV encodes a CC chemokine homolog (MCK-2). Human CMV UL146 encodes a neutrophil-attracting chemokine denoted viral CXC chemokine-1 (vCXCL1) that is as potent as host IL-8 and functions via the CXCR2 receptor, one of two human IL-8 receptors. Murine CMV MCK-2 is composed of a chemokine domain derived from open reading frame (ORF) m131 (and denoted MCK-1) as well as a domain derived from m129 that does not have sequence similarity to any known class of proteins. A synthetic version of murine CMV m131 (MCK-1) protein carries out many of the activities of a positive-acting chemokine, including transient release of intracellular calcium stores and cell adhesion of peritoneal macrophage populations. In the context of the viral genome and infection of the mouse host, the m131-m129 (MCK-2) gene product confers increased inflammation, higher levels of viremia, and higher titers of virus in salivary glands, consistent with a role in promoting dissemination by attracting an important mononuclear leukocyte population. Other characterized primate CMVs, but not other primate betaherpesviruses, encode gene products similar to human UL146 and UL147. Other characterized rodent CMVs encode a gene product similar to the murine CMV chemokine homolog, although not as a spliced gene product. Thus chemokines, like viral proteins that downmodulate MHC class I expression or have sequence homology to host MHC class I proteins, have evolved in primate and rodent CMVs to carry out an analogous set of immunomodulatory functions during infection of the host even though they arise from distinct origins.

  20. Impact of homologous and non-homologous recombination in the genomic evolution of Escherichia coli.

    PubMed

    Didelot, Xavier; Méric, Guillaume; Falush, Daniel; Darling, Aaron E

    2012-06-19

    Escherichia coli is an important species of bacteria that can live as a harmless inhabitant of the guts of many animals, as a pathogen causing life-threatening conditions or freely in the non-host environment. This diversity of lifestyles has made it a particular focus of interest for studies of genetic variation, mainly with the aim to understand how a commensal can become a deadly pathogen. Many whole genomes of E. coli have been fully sequenced in the past few years, which offer helpful data to help understand how this important species evolved. We compared 27 whole genomes encompassing four phylogroups of Escherichia coli (A, B1, B2 and E). From the core-genome we established the clonal relationships between the isolates as well as the role played by homologous recombination during their evolution from a common ancestor. We found strong evidence for sexual isolation between three lineages (A+B1, B2, E), which could be explained by the ecological structuring of E. coli and may represent on-going speciation. We identified three hotspots of homologous recombination, one of which had not been previously described and contains the aroC gene, involved in the essential shikimate metabolic pathway. We also described the role played by non-homologous recombination in the pan-genome, and showed that this process was highly heterogeneous. Our analyses revealed in particular that the genomes of three enterohaemorrhagic (EHEC) strains within phylogroup B1 have converged from originally separate backgrounds as a result of both homologous and non-homologous recombination. Recombination is an important force shaping the genomic evolution and diversification of E. coli, both by replacing fragments of genes with an homologous sequence and also by introducing new genes. In this study, several non-random patterns of these events were identified which correlated with important changes in the lifestyle of the bacteria, and therefore provide additional evidence to explain the

  1. Identification of plant microRNA homologs.

    PubMed

    Dezulian, Tobias; Remmert, Michael; Palatnik, Javier F; Weigel, Detlef; Huson, Daniel H

    2006-02-01

    MicroRNAs (miRNAs) are a recently discovered class of non-coding RNAs that regulate gene and protein expression in plants and animals. MiRNAs have so far been identified mostly by specific cloning of small RNA molecules, complemented by computational methods. We present a computational identification approach that is able to identify candidate miRNA homologs in any set of sequences, given a query miRNA. The approach is based on a sequence similarity search step followed by a set of structural filters.

  2. Genetic Homologies Among Streptomyces violaceoruber Strains

    PubMed Central

    Monson, A. M.; Bradley, S. G.; Enquist, L. W.; Cruces, Griselda

    1969-01-01

    Most of the genetic studies on streptomycetes have been done with cultures erroneously designated as Streptomyces coelicolor. To determine whether these cultures are genetically homologous with the S. violaceoruber nominifer, their deoxyribonucleic acids (DNA) were analyzed, and selected pairs of mutants were crossed. The four cultures used in genetic studies, and called S. coelicolor in the literature, were found to constitute a genospecies, based upon DNA hybridization and recombination tests. In addition, DNA from Actinopycnidium caeruleum formed extensive duplexes with S. violaceoruber DNA. S. violaceoruber cultures and A. caeruleum were distinctly different from the S. coelicolor nominifer. PMID:5370275

  3. Railway vehicle performance optimisation using virtual homologation

    NASA Astrophysics Data System (ADS)

    Magalhães, H.; Madeira, J. F. A.; Ambrósio, J.; Pombo, J.

    2016-09-01

    Unlike regular automotive vehicles, which are designed to travel in different types of roads, railway vehicles travel mostly in the same route during their life cycle. To accept the operation of a railway vehicle in a particular network, a homologation process is required according to local standard regulations. In Europe, the standards EN 14363 and UIC 518, which are used for railway vehicle acceptance, require on-track tests and/or numerical simulations. An important advantage of using virtual