WorldWideScience

Sample records for anthracis dna primase

  1. Preferential binding of DNA primase to the nuclear matrix

    International Nuclear Information System (INIS)

    Wood, S.H.; Collins, J.M.

    1986-01-01

    Several lines of research have stimulated interest in the nuclear matrix as the subcellular site of DNA replication. The authors have recently reported a relationship between rates of DNA synthesis and the differential binding of polymerase α to the nuclear matrix. They now report the detection of DNA primase bound to the HeLa nuclear matrix. Matrix-bound primase can be measured either indirectly, by the incorporation of [ 32 P] dAMP into an unprimed single-stranded template, or directly, by the incorporation of [ 3 H] AMP into matrix DNA. Characteristics of this system include a requirement for ATP, inhibition by adenosine-5'-0-(3'-thiotriphosphate), a primase inhibitor, and insensitivity to aphidicolin and α-amanitine, inhibitors of polymerase α and RNA polymerase, respectively. Subcellular quantification of primase and polymerase α activity revealed that while a majority of primase activity is bound to the matrix (72%), only 32% of polymerase α activity is matrix-bound. Treatment of the nuclear matrix with β-D-Octylglucoside allowed the solubilization of 54% of primase activity and 39% of polymerase α activity. This data provides further evidence of a structural and functional role for the nuclear matrix in DNA replication. The ability to solubilize matrix-bound replicative enzymes may prove to be an important tool in the elucidation of the spatial organization of DNA replication

  2. Identification and characterization of a DNA primase activity present in herpes simplex virus type 1-infected HeLa cells

    International Nuclear Information System (INIS)

    Holmes, A.M.; Wietstock, S.M.; Ruyechan, W.T.

    1988-01-01

    A novel DNA primase activity has been identified in HeLa cells infected with herpes simplex virus type 1 (HSV-1). Such an activity has not been detected in mock-infected cells. The primase activity coeluted with a portion of HSV-1 DNA polymerase from single-stranded DNA agarose columns loaded with high-salt extracts derived from infected cells. This DNA primase activity could be distinguished from host HeLa cell DNA primase by several criteria. First, the pH optimum of the HSV primase was relatively broad and peaked at 8.2 to 8.7 pH units. Second, freshly isolated HSV DNA primase was less salt sensitive than the HeLa primase. Third, antibodies raised against individual peptides of the calf thymus DNA polymerase:primase complex cross-reacted with the HeLa primase but did not react with the HSV DNA primase. Fourth, freshly prepared HSV DNA primase appeared to be associated with the HSV polymerase, but after storage at 4 degree C for several weeks, the DNA primase separated from the viral DNA polymerase. This free DNA primase had an apparent molecular size of approximately 40 kilodaltons, whereas free HeLa DNA primase had an apparent molecular size of approximately 110 kilodaltons. On the basis of these data, the authors believe that the novel DNA primase activity in HSV-infected cells may be virus coded and that this enzyme represents a new and important function involved in the replication of HSV DNA

  3. Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

    KAUST Repository

    Lee, Seung-Joo; Zhu, Bin; Hamdan, Samir; Richardson, Charles C.

    2010-01-01

    DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical

  4. Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

    KAUST Repository

    Lee, Seung-Joo

    2010-03-28

    DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5\\'-TGGTC-3\\') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain. The Author(s) 2010. Published by Oxford University Press.

  5. DNA Recognition by the DNA Primase of Bacteriophage T7: A Structure Function Study of the Zinc-Binding Domain

    International Nuclear Information System (INIS)

    Akabayov, B.; Lee, S.; Akabayov, S.; Rekhi, S.; Zhu, B.; Richardson, C.

    2009-01-01

    Synthesis of oligoribonucleotide primers for lagging-strand DNA synthesis in the DNA replication system of bacteriophage T7 is catalyzed by the primase domain of the gene 4 helicase-primase. The primase consists of a zinc-binding domain (ZBD) and an RNA polymerase (RPD) domain. The ZBD is responsible for recognition of a specific sequence in the ssDNA template whereas catalytic activity resides in the RPD. The ZBD contains a zinc ion coordinated with four cysteine residues. We have examined the ligation state of the zinc ion by X-ray absorption spectroscopy and biochemical analysis of genetically altered primases. The ZBD of primase engaged in catalysis exhibits considerable asymmetry in coordination to zinc, as evidenced by a gradual increase in electron density of the zinc together with elongation of the zinc-sulfur bonds. Both wild-type primase and primase reconstituted from purified ZBD and RPD have a similar electronic change in the level of the zinc ion as well as the configuration of the ZBD. Single amino acid replacements in the ZBD (H33A and C36S) result in the loss of both zinc binding and its structural integrity. Thus the zinc in the ZBD may act as a charge modulation indicator for the surrounding sulfur atoms necessary for recognition of specific DNA sequences.

  6. The Mini-Chromosome Maintenance (Mcm) Complexes Interact with DNA Polymerase α-Primase and Stimulate Its Ability to Synthesize RNA Primers

    Science.gov (United States)

    You, Zhiying; De Falco, Mariarosaria; Kamada, Katsuhiko; Pisani, Francesca M.; Masai, Hisao

    2013-01-01

    The Mini-chromosome maintenance (Mcm) proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2∼7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2∼7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes. PMID:23977294

  7. The mini-chromosome maintenance (Mcm complexes interact with DNA polymerase α-primase and stimulate its ability to synthesize RNA primers.

    Directory of Open Access Journals (Sweden)

    Zhiying You

    Full Text Available The Mini-chromosome maintenance (Mcm proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2~7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2~7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes.

  8. Mechanisms by Which Human DNA Primase Chooses To Polymerize a Nucleoside Triphosphate

    Czech Academy of Sciences Publication Activity Database

    Urban, M.; Joubert, Nicolas; Purse, B. W.; Hocek, Michal; Kuchta, R. D.

    2010-01-01

    Roč. 49, č. 4 (2010), s. 727-735 ISSN 0006-2960 R&D Projects: GA MŠk LC512; GA AV ČR IAA400550902 Grant - others:NIH(US) GM54194 Institutional research plan: CEZ:AV0Z40550506 Keywords : C-nucleosides * DNA polymerase * primase * mechanism Subject RIV: CC - Organic Chemistry Impact factor: 3.226, year: 2010

  9. Human DNA primase uses Watson-Crick hydrogen bonds to distinguish between correct and incorrect nucleoside triphosphates.

    Science.gov (United States)

    Moore, Chad L; Zivkovic, Aleksandra; Engels, Joachim W; Kuchta, Robert D

    2004-09-28

    Human DNA primase synthesizes short RNA primers that DNA polymerase alpha further elongates. Primase readily misincorporates the natural NTPs and will generate a wide variety of mismatches. In contrast, primase exhibited a remarkable resistance to polymerizing NTPs containing unnatural bases. This included bases whose shape was almost identical to the natural bases (4-aminobenzimidazole and 4,6-difluorobenzimidazole), bases shaped very differently than a natural base [e.g., 5- and 6-(trifluoromethyl)benzimidazole], bases much more hydrophobic than a natural base [e.g., 4- and 7-(trifluoromethyl)benzimidazole], bases of similar hydrophobicity as a natural base but with the Watson-Crick hydrogen-bonding groups in unusual positions (7-beta-D-guanine), and bases capable of forming only one Watson-Crick hydrogen bond with the template base (purine and 4-aminobenzimidazole). Primase only polymerized NTP analogues containing bases capable of forming hydrogen bonds between the equivalent of both N-1 and the exocyclic group at C-6 of a purine NTP (2-fluoroadenine, 2-chloroadenine, 3-deazaadenine, and hypoxanthine) and N-3 and the exocyclic group at C-4 of a pyrimidine. These data indicate that human primase requires the formation of Watson-Crick hydrogen bonds in order to polymerize a NTP, a situation very different than what is observed with some DNA polymerases. The implications of these results with respect to current theories of how polymerases discriminate between right and wrong (d)NTPs are discussed.

  10. Three-dimensional structure of N-terminal domain of DnaB helicase and helicase-primase interactions in Helicobacter pylori.

    Directory of Open Access Journals (Sweden)

    Tara Kashav

    2009-10-01

    Full Text Available Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD of H. pylori DnaB (HpDnaB helicase at 2.2 A resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.

  11. Ap/sub 4/A interactions with a multiprotein form of DNA polymerase. cap alpha. - primase from HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Baril, E F; Owen, M W; Vishwanatha, J K; Zamecnik, P C

    1984-06-01

    In previous studies, it was shown that Ap/sub 4/A can function as a primer for in vitro DNA synthesis by the multiprotein form of DNA polymerase ..cap alpha.. with single-stranded DNA and an octadecamer double-stranded DNA template. In these studies, the authors show that Ap/sub 4/A that is greater than 99% pure by high performance liquid chromatography also stimulates the incorporation of (..cap alpha../sup 32/P)ATP into the 10-15 oligoribonucleotide primer with poly(dT) template by the primase that is resolved from the polymerase ..cap alpha.. core enzyme. Other dinucleotides or dinucleotide polyphosphates (e.g. ApA, Ap/sub 2/A or Ap/sub 3/A) do not enhance the incorporation of (..cap alpha../sup 32/P)ATP in this reaction. The results from phosphate transfer experiments demonstrate a covalent linkage between (/sup 3/H)Ap/sub 4/A and the /sup 32/P-labeled oligoriboadenylate that is synthesized by the primase.

  12. Solid-state NMR chemical-shift perturbations indicate domain reorientation of the DnaG primase in the primosome of Helicobacter pylori

    Energy Technology Data Exchange (ETDEWEB)

    Gardiennet, Carole [Université de Lorraine, CNRS, CRM2, UMR 7036 (France); Wiegand, Thomas [ETH Zurich, Physical Chemistry (Switzerland); Bazin, Alexandre [Université de Lyon 1, Molecular Microbiology and Structural Biochemistry, Labex Ecofect, UMR 5086 CNRS (France); Cadalbert, Riccardo [ETH Zurich, Physical Chemistry (Switzerland); Kunert, Britta; Lacabanne, Denis [Université de Lyon 1, Molecular Microbiology and Structural Biochemistry, Labex Ecofect, UMR 5086 CNRS (France); Gutsche, Irina [Université Grenoble Alpes, Institut de Biologie Structurale (IBS), CNRS, IBS, CEA, IBS (France); Terradot, Laurent, E-mail: l.terradot@ibcp.fr [Université de Lyon 1, Molecular Microbiology and Structural Biochemistry, Labex Ecofect, UMR 5086 CNRS (France); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); Böckmann, Anja, E-mail: a.bockmann@ibcp.fr [Université de Lyon 1, Molecular Microbiology and Structural Biochemistry, Labex Ecofect, UMR 5086 CNRS (France)

    2016-03-15

    We here investigate the interactions between the DnaB helicase and the C-terminal domain of the corresponding DnaG primase of Helicobacter pylori using solid-state NMR. The difficult crystallization of this 387 kDa complex, where the two proteins interact in a six to three ratio, is circumvented by simple co-sedimentation of the two proteins directly into the MAS-NMR rotor. While the amount of information that can be extracted from such a large protein is still limited, we can assign a number of amino-acid residues experiencing significant chemical-shift perturbations upon helicase-primase complex formation. The location of these residues is used as a guide to model the interaction interface between the two proteins in the complex. Chemical-shift perturbations also reveal changes at the interaction interfaces of the hexameric HpDnaB assembly on HpDnaG binding. A structural model of the complex that explains the experimental findings is obtained.

  13. The UL5 and UL52 subunits of the herpes simplex virus type 1 helicase-primase subcomplex exhibit a complex interdependence for DNA binding.

    Science.gov (United States)

    Biswas, N; Weller, S K

    2001-05-18

    Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. The UL5 protein contains seven motifs found in all members of helicase Superfamily 1 (SF1), and the UL52 protein contains several conserved motifs found in primases; however, the contributions of each subunit to the biochemical activities of the subcomplex are not clear. In this work, the DNA binding properties of wild type and mutant subcomplexes were examined using single-stranded, duplex, and forked substrates. A gel mobility shift assay indicated that the UL5-UL52 subcomplex binds more efficiently to the forked substrate than to either single strand or duplex DNA. Although nucleotides are not absolutely required for DNA binding, ADP stimulated the binding of UL5-UL52 to single strand DNA whereas ATP, ADP, and adenosine 5'-O-(thiotriphosphate) stimulated the binding to a forked substrate. We have previously shown that both subunits contact single-stranded DNA in a photocross-linking assay (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076). In this study, photocross-linking assays with forked substrates indicate that the UL5 and UL52 subunits contact the forked substrates at different positions, UL52 at the single-stranded DNA tail and UL5 near the junction between single-stranded and double-stranded DNA. Neither subunit was able to cross-link a forked substrate when 5-iododeoxyuridine was located within the duplex portion. Photocross-linking experiments with subcomplexes containing mutant versions of UL5 and wild type UL52 indicated that the integrity of the ATP binding region is important for DNA binding of both subunits. These results support our previous proposal that UL5 and UL52 exhibit a complex interdependence for DNA binding (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076) and indicate that the UL52 subunit may play a more active role in helicase activity than had previously been

  14. Identification of conserved amino acids in the herpes simplex virus type 1 UL8 protein required for DNA synthesis and UL52 primase interaction in the virus replisome.

    Science.gov (United States)

    Muylaert, Isabella; Zhao, Zhiyuan; Andersson, Torbjörn; Elias, Per

    2012-09-28

    We have used oriS-dependent transient replication assays to search for species-specific interactions within the herpes simplex virus replisome. Hybrid replisomes derived from herpes simplex virus type 1 (HSV-1) and equine herpesvirus type 1 (EHV-1) failed to support DNA replication in cells. Moreover, the replisomes showed a preference for their cognate origin of replication. The results demonstrate that the herpesvirus replisome behaves as a molecular machine relying on functionally important interactions. We then searched for functional interactions in the replisome context by subjecting HSV-1 UL8 protein to extensive mutagenesis. 52 mutants were made by replacing single or clustered charged amino acids with alanines. Four mutants showed severe replication defects. Mutant A23 exhibited a lethal phenotype, and mutants A49, A52 and A53 had temperature-sensitive phenotypes. Mutants A49 and A53 did not interact with UL52 primase as determined by co-immunoprecipitation experiments. Using GFP-tagged UL8, we demonstrate that all mutants were unable to support formation of ICP8-containing nuclear replication foci. Extended mutagenesis suggested that a highly conserved motif corresponding to mutant A49 serves an important role for establishing a physical contact between UL8 and UL52. The replication-defective mutations affected conserved amino acids, and similar phenotypes were observed when the corresponding mutations were introduced into EHV-1 UL8.

  15. The role of DNA restriction-modification systems in the biology of Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Ramakrishnan eSitaraman

    2016-01-01

    Full Text Available Restriction-modification (R-M systems are widespread among prokaryotes and, depending on their type, may be viewed as selfish genetic elements that persist as toxin-antitoxin modules or as cellular defense systems against phage infection. Studies in the last decade have made it amply clear that these two options do not exhaust the list of possible biological roles for R-M systems. Their presence in a cell may also have a bearing on other processes such as horizontal gene transfer and gene regulation. From genome sequencing and experimental data, we know that Bacillus anthracis encodes at least three methylation-dependent (typeIV restriction endonucleases, and an orphan DNA methyltransferase. In this article, we first present an outline of our current knowledge of R-M systems in Bacillus anthracis. Based on available DNA sequence data, and on our current understanding of the functions of similar genes in other systems, we conclude with hypotheses on the possible roles of the three restriction endonucleases and the orphan DNA methyltransferase.

  16. Literature Review of DNA-Based Subspecies Analysis of Bacillus Anthracis Burkholderia Pseudomallel Burkholderia Mallei, and Yersinia Pestis

    National Research Council Canada - National Science Library

    Harvey, Steven

    1999-01-01

    ...; Bacillus anthracis, Burkholderia pseudomallei, Burkholderia mallei, and Yersinia pestis. Considerable research has been accomplished for the identification of polymorphisms from the strains B. anthracis and B. pseudomallei. The B...

  17. Measurements of DNA Damage and Repair in Bacillus anthracis Sterne Spores by UV Radiation

    Science.gov (United States)

    2014-09-18

    tightly into chromosomal structure, which serves to protect the DNA. The DNA is coiled several times and wrapped tightly around proteins. The wrapping...helps the DNA keep its chromatin structure. When DNA is to be replicated, enzymes unwind the chromosomal DNA. Therefore, DNA is not specifically used...2 See Appendix for Plasmid Information sheet. 30 the ice crystals disappeared . The tube was gently mixed by hand

  18. The mechanism of DNA ejection in the Bacillus anthracis spore-binding phage 8a revealed by cryo-electron tomography

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Xiaofeng [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States); Walter, Michael H. [Department of Biology, University of Northern Iowa, Cedar Falls, IA 50614 (United States); Paredes, Angel [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States); Morais, Marc C., E-mail: mcmorais@utmb.edu [Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555 (United States); Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555 (United States); Liu, Jun, E-mail: Jun.Liu.1@uth.tmc.edu [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States)

    2011-12-20

    The structure of the Bacillus anthracis spore-binding phage 8a was determined by cryo-electron tomography. The phage capsid forms a T = 16 icosahedron attached to a contractile tail via a head-tail connector protein. The tail consists of a six-start helical sheath surrounding a central tail tube, and a structurally novel baseplate at the distal end of the tail that recognizes and attaches to host cells. The parameters of the icosahedral capsid lattice and the helical tail sheath suggest protein folds for the capsid and tail-sheath proteins, respectively, and indicate evolutionary relationships to other dsDNA viruses. Analysis of 2518 intact phage particles show four distinct conformations that likely correspond to four sequential states of the DNA ejection process during infection. Comparison of the four observed conformations suggests a mechanism for DNA ejection, including the molecular basis underlying coordination of tail sheath contraction and genome release from the capsid.

  19. Overexpression, crystallization and preliminary X-ray crystallographic analysis of the RNA polymerase domain of primase from Streptococcus mutans strain UA159

    International Nuclear Information System (INIS)

    Im, Dong-Won; Kim, Tae-O; Jung, Ha Yun; Oh, Ji Eun; Lee, Se Jin; Heo, Yong-Seok

    2011-01-01

    The RNA polymerase domain of primase from S. mutans strain UA159 was cloned, overexpressed, purified and crystallized. X-ray diffraction data were collected to a resolution of 1.60 Å. Primase is the enzyme that synthesizes RNA primers on single-stranded DNA during normal DNA replication. In this study, the catalytic core domain of primase from Streptococcus mutans UA159 was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 1.60 Å resolution using a synchrotron-radiation source. The crystal belonged to space group P4 1 or P4 3 , with unit-cell parameters a = b = 52.63, c = 110.31 Å. The asymmetric unit is likely to contain one molecule, with a corresponding V M of 1.77 Å 3 Da −1 and a solvent content of 30.7%

  20. Double-strand break repair and genetic recombination in topoisomerase and primase mutants of bacteriophage T4.

    Science.gov (United States)

    Shcherbakov, Victor P; Kudryashova, Elena

    2014-09-01

    -branch migration step of the DSB repair pathway and partially deficient in HJ initiation. In apparent contradiction to their effects on the DSB-induced site-specific recombination, the topoisomerase and primase mutants demonstrated about 3-8-fold increase in the recombinant frequencies in the ordinary crosses, with the recombination running exclusively via patches. This implies that most of the spontaneous recombination events are not initiated by dsDNA ends in these mutants. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Mutations in the putative zinc-binding motif of UL52 demonstrate a complex interdependence between the UL5 and UL52 subunits of the human herpes simplex virus type 1 helicase/primase complex.

    Science.gov (United States)

    Chen, Yan; Carrington-Lawrence, Stacy D; Bai, Ping; Weller, Sandra K

    2005-07-01

    Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface.

  2. Antiviral drug resistance and helicase-primase inhibitors of herpes simplex virus.

    Science.gov (United States)

    Field, Hugh J; Biswas, Subhajit

    2011-02-01

    A new class of chemical inhibitors has been discovered that interferes with the process of herpesvirus DNA replication. To date, the majority of useful herpesvirus antivirals are nucleoside analogues that block herpesvirus DNA replication by targeting the DNA polymerase. The new helicase-primase inhibitors (HPI) target a different enzyme complex that is also essential for herpesvirus DNA replication. This review will place the HPI in the context of previous work on the nucleoside analogues. Several promising highly potent HPI will be described with a particular focus on the identification of drug-resistance mutations. Several HPI have good pharmacological profiles and are now at the outset of phase II clinical trials. Provided there are no safety issues to stop their progress, this new class of compound will be a major advance in the herpesvirus antiviral field. Furthermore, HPI are likely to have a major impact on the therapy and prevention of herpes simplex virus and varicella zoster in both immunocompetent and immunocompromised patients alone or in combination with current nucleoside analogues. The possibility of acquired drug-resistance to HPI will then become an issue of great practical importance. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species.

    Directory of Open Access Journals (Sweden)

    Hirohito Ogawa

    Full Text Available Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.

  4. Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization

    Directory of Open Access Journals (Sweden)

    Redkar Rajendra

    2004-02-01

    Full Text Available Abstract Background Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH was used to identify specific chromosomal sequences unique to B. anthracis. Results Two SSH libraries were generated. Genomic DNA from plasmid-cured B. anthracis was used as the tester DNA in both libraries, while genomic DNA from either B. cereus or B. thuringiensis served as the driver DNA. Progressive screening of the libraries by colony filter and Southern blot analyses identified 29 different clones that were specific for the B. anthracis chromosome relative not only to the respective driver DNAs, but also to seven other different strains of B. cereus and B. thuringiensis included in the process. The nucleotide sequences of the clones were compared with those found in genomic databases, revealing that over half of the clones were located into 2 regions on the B. anthracis chromosome. Conclusions Genes encoding potential cell wall synthesis proteins dominated one region, while bacteriophage-related sequences dominated the other region. The latter supports the hypothesis that acquisition of these bacteriophage sequences occurred during or after speciation of B. anthracis relative to B. cereus and B. thuringiensis. This study provides insight into the chromosomal differences between B. anthracis and its closest phylogenetic relatives.

  5. Novel Sample Preparation Method for Safe and Rapid Detection of Bacillus anthracis Spores in Environmental Powders and Nasal Swabs

    OpenAIRE

    Luna, Vicki A.; King, Debra; Davis, Carisa; Rycerz, Tony; Ewert, Matthew; Cannons, Andrew; Amuso, Philip; Cattani, Jacqueline

    2003-01-01

    Bacillus anthracis spores have been used as a biological weapon in the United States. We wanted to develop a safe, rapid method of sample preparation that provided safe DNA for the detection of spores in environmental and clinical specimens. Our method reproducibly detects B. anthracis in samples containing

  6. Helicase-primase inhibitor amenamevir for herpesvirus infection: Towards practical application for treating herpes zoster.

    Science.gov (United States)

    Shiraki, K

    2017-11-01

    Valacyclovir and famciclovir enabled successful systemic therapy for treating herpes simplex virus (HSV) and varicella zoster virus (VZV) infection by their phosphorylation with viral thymidine kinase. Helicase-primase inhibitors (HPIs) inhibit the progression of the replication fork, an initial step in DNA synthesis to separate the double strand into two single strands. The HPIs amenamevir and pritelivir have a novel mechanism of action, once-daily administration with nonrenal excretory characteristics, and clinical efficacy for genital herpes. Amenamevir exhibits anti-VZV and anti-HSV activity while pritelivir only has anti-HSV activity. A clinical trial of amenamevir for herpes zoster has been completed, and amenamevir has been licensed and successfully used in 20,000 patients with herpes zoster so far in Japan. We have characterized the features of the antiviral action of amenamevir and, unlike acyclovir, the drug's antiviral activity is not influenced by the viral replication cycle. Amenamevir is opening a new era of antiherpes therapy. Copyright 2017 Clarivate Analytics.

  7. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    DEFF Research Database (Denmark)

    Ågren, Joakim; Hamidjaja, Raditijo A.; Hansen, Trine

    2013-01-01

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely...... on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S...... targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European...

  8. Detection of Bacillus anthracis in the air, soil and animal tissue

    Directory of Open Access Journals (Sweden)

    Kušar D.

    2012-01-01

    Full Text Available The objective of the present work was to establish effective and rapid diagnostic methods for the detection of Bacillus anthracis, a highly virulent zoonotic pathogen, in the air, soil and animal (or human tissue samples. Liquid culture of B. anthracis was aerosolized and four air sampling procedures were employed. Detection of B. anthracis in the air samples was successful with RCS High Flow sampler (culturebased detection and when sampling through the air filter (molecular detection using SmartHelix Complex Samples DNA Extraction Kit. Liquid B. anthracis culture was also employed for spiking the homogenised bovine lymphatic gland tissue and soil samples. DNA extraction was performed using three different commercial kits for each sample type. High Pure PCR Template Preparation Kit was the most effective for DNA extraction from animal tissue samples. Detection in the soil was successful when PowerSoil DNA Isolation Kit was used. Our results indicate that B. anthracis can be monitored in different matrices by rapid molecular methods when appropriate sampling and DNA extraction procedures are employed prior to PCR assay. The selected rapid protocols can be implemented in specialized veterinary or human diagnostic laboratories with moderate costs.

  9. The Bacillus anthracis chromosome contains four conserved, excision-proficient, putative prophages

    Directory of Open Access Journals (Sweden)

    Sozhamannan Shanmuga

    2006-04-01

    Full Text Available Abstract Background Bacillus anthracis is considered to be a recently emerged clone within the Bacillus cereus sensu lato group. The B. anthracis genome sequence contains four putative lambdoid prophages. We undertook this study in order to understand whether the four prophages are unique to B. anthracis and whether they produce active phages. Results More than 300 geographically and temporally divergent isolates of B. anthracis and its near neighbors were screened by PCR for the presence of specific DNA sequences from each prophage region. Every isolate of B. anthracis screened by PCR was found to produce all four phage-specific amplicons whereas none of the non-B. anthracis isolates, produced more than one phage-specific amplicon. Excision of prophages could be detected by a PCR based assay for attP sites on extra-chromosomal phage circles and for attB sites on phage-excised chromosomes. SYBR-green real-time PCR assays indicated that prophage excision occurs at very low frequencies (2 × 10-5 - 8 × 10-8/cell. Induction with mitomycin C increased the frequency of excision of one of the prophages by approximately 250 fold. All four prophages appear to be defective since, mitomycin C induced culture did not release any viable phage particle or lyse the cells or reveal any phage particle under electron microscopic examination. Conclusion The retention of all four putative prophage regions across all tested strains of B. anthracis is further evidence of the very recent emergence of this lineage and the prophage regions may be useful for differentiating the B. anthracis chromosome from that of its neighbors. All four prophages can excise at low frequencies, but are apparently defective in phage production.

  10. Decontamination of Soil Contaminated with Bacillus anthracis ...

    Science.gov (United States)

    Technical Brief This technical summary will provide decontamination personnel rapid access to information on which decontamination approaches are most effective for soils contaminated with B anthracis.

  11. Characterization of 21 Strains of Bacillus Anthracis

    National Research Council Canada - National Science Library

    Kournikakis, B

    2000-01-01

    Twenty-one strains of Bacillus anthracis currently held in the culture collection at DRES were characterized by colonial morphology, antibiotic sensitivity and BiologTM metabolic identification profiles...

  12. Improvements to a Markerless Allelic Exchange System for Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Roger D Plaut

    Full Text Available A system was previously developed for conducting I-SceI-mediated allelic exchange in Bacillus anthracis. In this system, recombinational loss of a chromosomally-integrated allelic exchange vector is stimulated by creation of a double-stranded break within the vector by the homing endonuclease I-SceI. Although this system is reasonably efficient and represents an improvement in the tools available for allelic exchange in B. anthracis, researchers are nonetheless required to "pick and patch" colonies in order to identify candidate "exchangeants." In the present study, a number of improvements have been made to this system: 1 an improved I-SceI-producing plasmid includes oriT so that both plasmids can now be introduced by conjugation, thus avoiding the need for preparing electro-competent cells of each integration intermediate; 2 antibiotic markers have been changed to allow the use of the system in select agent strains; and 3 both plasmids have been marked with fluorescent proteins, allowing the visualization of plasmid segregation on a plate and obviating the need for "picking and patching." These modifications have made the process easier, faster, and more efficient, allowing for parallel construction of larger numbers of mutant strains. Using this improved system, the genes encoding the tripartite anthrax toxin were deleted singly and in combination from plasmid pXO1 of Sterne strain 34F2. In the course of this study, we determined that DNA transfer to B. anthracis could be accomplished by conjugation directly from a methylation-competent E. coli strain.

  13. Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: primase as the sole priming enzyme

    NARCIS (Netherlands)

    van der Ende, A.; Baker, T. A.; Ogawa, T.; Kornberg, A.

    1985-01-01

    The enzymatic replication of plasmids containing the unique (245 base pair) origin of the Escherichia coli chromosome (oriC) can be initiated with any of three enzyme priming systems: primase alone, RNA polymerase alone, or both combined (Ogawa, T., Baker, T. A., van der Ende, A. & Kornberg, A.

  14. Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis

    Science.gov (United States)

    2011-09-01

    future predictive modeling toolkits. 1 1. Introduction The use of Bacillus anthracis as a bio - weapon in the United States in 2001 affirmed the need...for improved sensing and detection of biological weapons of mass destruction (WMD). Protective Antigen (PA) protein of Bacillus anthracis is the...Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis by Deborah A. Sarkes, Joshua M. Kogot, Irene Val-Addo

  15. Herpes Simplex Virus-1 DNA Primase: A Remarkably Inaccurate yet Selective Polymerase

    Czech Academy of Sciences Publication Activity Database

    Urban, M.; Joubert, Nicolas; Hocek, Michal; Alexander, R. E.; Kuchta, R. D.

    2009-01-01

    Roč. 48, č. 46 (2009), s. 10866-10881 ISSN 0006-2960 R&D Projects: GA MŠk LC512; GA AV ČR IAA400550902 Grant - others:NIH(US) AI059764 Institutional research plan: CEZ:AV0Z40550506 Keywords : HSV-1 * herpes simplex virus-1 * Pyr * pyrimidine Subject RIV: CC - Organic Chemistry Impact factor: 3.226, year: 2009

  16. Eukaryotic DNA Replicases

    KAUST Repository

    Zaher, Manal S.; Oke, Muse; Hamdan, Samir

    2014-01-01

    The current model of the eukaryotic DNA replication fork includes three replicative DNA polymerases, polymerase α/primase complex (Pol α), polymerase δ (Pol δ), and polymerase ε (Pol ε). The primase synthesizes 8–12 nucleotide RNA primers that are extended by the DNA polymerization activity of Pol α into 30–35 nucleotide RNA-DNA primers. Replication factor C (RFC) opens the polymerase clamp-like processivity factor, proliferating cell nuclear antigen (PCNA), and loads it onto the primer-template. Pol δ utilizes PCNA to mediate highly processive DNA synthesis, while Pol ε has intrinsic high processivity that is modestly stimulated by PCNA. Pol ε replicates the leading strand and Pol δ replicates the lagging strand in a division of labor that is not strict. The three polymerases are comprised of multiple subunits and share unifying features in their large catalytic and B subunits. The remaining subunits are evolutionarily not related and perform diverse functions. The catalytic subunits are members of family B, which are distinguished by their larger sizes due to inserts in their N- and C-terminal regions. The sizes of these inserts vary among the three polymerases, and their functions remain largely unknown. Strikingly, the quaternary structures of Pol α, Pol δ, and Pol ε are arranged similarly. The catalytic subunits adopt a globular structure that is linked via its conserved C-terminal region to the B subunit. The remaining subunits are linked to the catalytic and B subunits in a highly flexible manner.

  17. Eukaryotic DNA Replicases

    KAUST Repository

    Zaher, Manal S.

    2014-11-21

    The current model of the eukaryotic DNA replication fork includes three replicative DNA polymerases, polymerase α/primase complex (Pol α), polymerase δ (Pol δ), and polymerase ε (Pol ε). The primase synthesizes 8–12 nucleotide RNA primers that are extended by the DNA polymerization activity of Pol α into 30–35 nucleotide RNA-DNA primers. Replication factor C (RFC) opens the polymerase clamp-like processivity factor, proliferating cell nuclear antigen (PCNA), and loads it onto the primer-template. Pol δ utilizes PCNA to mediate highly processive DNA synthesis, while Pol ε has intrinsic high processivity that is modestly stimulated by PCNA. Pol ε replicates the leading strand and Pol δ replicates the lagging strand in a division of labor that is not strict. The three polymerases are comprised of multiple subunits and share unifying features in their large catalytic and B subunits. The remaining subunits are evolutionarily not related and perform diverse functions. The catalytic subunits are members of family B, which are distinguished by their larger sizes due to inserts in their N- and C-terminal regions. The sizes of these inserts vary among the three polymerases, and their functions remain largely unknown. Strikingly, the quaternary structures of Pol α, Pol δ, and Pol ε are arranged similarly. The catalytic subunits adopt a globular structure that is linked via its conserved C-terminal region to the B subunit. The remaining subunits are linked to the catalytic and B subunits in a highly flexible manner.

  18. Sample collection of virulent and non-virulent B. anthracis and Y. pestis for bioforensics analysis

    Energy Technology Data Exchange (ETDEWEB)

    Hong-geller, Elizabeth [Los Alamos National Laboratory; Valdez, Yolanda E [Los Alamos National Laboratory; Shou, Yulin [Los Alamos National Laboratory; Yoshida, Thomas M [Los Alamos National Laboratory; Marrone, Babetta L [Los Alamos National Laboratory; Dunbar, John [Los Alamos National Laboratory

    2009-01-01

    Validated sample collection methods are needed for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. To address this need, we evaluated the sample recovery efficiencies of two collection methods -- swabs and wipes -- for both non-virulent and virulent strains of B. anthracis and Y. pestis from four types of non-porous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using Real-time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface-dependent for virulent strains than non-virulent strains. For the two non-virulent strains, B. anthracis Sterne and Y. pestis A1122, collection efficiency was approximately 100% and 1 %, respectively, from all four surfaces. In contrast, recovery of B. anthracis Ames spores and Y. pestis C092 from vinyl and plastic was generally lower compared to collection from glass or stainless steel, suggesting that surface hydrophobicity may playa role in the strength of pathogen adhesion. The surface-dependent collection efficiencies observed with the virulent strains may arise from strain-specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. These findings contribute to validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.

  19. The Efficiency of Methionine as a Radioprotectant of Bacillus anthracis for Cell Viability and Outgrowth Time after UVC and Gamma Irradiation

    Science.gov (United States)

    2015-03-01

    Qiagen, "EndoFree Plasmid Purification Handbook ," 2012. [Online]. Available: http://www.qiagen.com/resources/resourcedetail?id=f8ed5bab-15c3-4211 - bfa8...115] L. J. Hoffman, "Thermogravimetric Analysis of Bacillus anthracis Spores of DNA by Spectroscopy and Chromatography of Pyrolysis Products," M.S

  20. Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

    OpenAIRE

    Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M.

    2014-01-01

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthesis operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity, and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-bin...

  1. Enzymatic recognition of DNA replication origins

    International Nuclear Information System (INIS)

    Stayton, M.M.; Bertsch, L.; Biswas, S.

    1983-01-01

    In this paper we discuss the process of recognition of the complementary-strand origin with emphasis on RNA polymerase action in priming M13 DNA replication, the role of primase in G4 DNA replication, and the function of protein n, a priming protein, during primosome assembly. These phage systems do not require several of the bacterial DNA replication enzymes, particularly those involved in the regulation of chromosome copy number of the initiatiion of replication of duplex DNA. 51 references, 13 figures, 1 table

  2. ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

    Science.gov (United States)

    Yang, Mu; Wang, Ganggang

    2016-09-15

    The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Identification of Proteins in the Exosporium of Bacillus Anthracis

    National Research Council Canada - National Science Library

    Redmond, Caroline; Baillie, Leslie W. J; Hibbs, Stephen; Moir, Arthur J. G; Moir, Anne

    2004-01-01

    .... The protein profiles of SDS-PAGE-separated exosporium extracts were similar for all three. This suggests that avirulent variants lacking either or both plasmids are realistic models for studying the exosporium from spores of B. anthracis...

  4. Dendritic Cells Endocytose Bacillus Anthracis Spores: Implications for Anthrax Pathogenesis

    National Research Council Canada - National Science Library

    Brittingham, Katherine C; Ruthel, Gordon; Panchal, Rekha G; Fuller, Claudette L; Ribot, Wilson J

    2005-01-01

    Phagocytosis of inhaled Bacillus anthracis spores and subsequent trafficking to lymph nodes are decisive events in the progression of inhaled anthrax because they initiate germination and dissemination of spores...

  5. Genetic Characterization of Bacillus anthracis 17 JB strain.

    Science.gov (United States)

    Seyed-Mohamadi, Sakineh; Moradi Bidhendi, Soheila; Tadayon, Keyvan; Ghaderi, Rainak

    2015-06-01

    Bacillus anthracis is one of the most homogenous bacteria ever described. Some level of diversity. Bacillus anthracis 17JB is a laboratory strain It is broadly used as a challenge strain in guinea pigs for potency test of anthrax vaccine. This work describes genetic characterization of B. anthracis 17 JB strain using the SNPs and MLVA genotyping. In SNPs typing, the originally French 17JB strain represented the A.Br. 008/009 subgroup. In Levy's genotyping method, 843, 451 and 864 bp long fragments were identified at AA03, AJ03 and AA07 loci, respectively. In the vaccine manufacturer perspective these findings are much valuable on their own account, but similar research is required to extend molecular knowledge of B. anthracis epidemiology in Persia.

  6. Functional Comparison of the Two Bacillus anthracis Glutamate Racemases▿

    OpenAIRE

    Dodd, Dylan; Reese, Joseph G.; Louer, Craig R.; Ballard, Jimmy D.; Spies, M. Ashley; Blanke, Steven R.

    2007-01-01

    Glutamate racemase activity in Bacillus anthracis is of significant interest with respect to chemotherapeutic drug design, because l-glutamate stereoisomerization to d-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of B. anthracis predicts two genes encoding glutamate racemases, racE1 and rac...

  7. Bacillus Collagen Like Protein of Anthracis: Immunological and Functional Analyses

    Science.gov (United States)

    2007-09-21

    factors such as heat, radiation, desiccation, pH extremes, and toxic chemicals (87). Cultivation of B. anthracis can be accomplished on non...obtained from the Naval Medical Research Center. B. anthracis was induced to sporulate on Leighton-Doi Medium (LD) (75). The broth was inoculated with an...heated at 65°C for 30 minutes, diluted, and plated on trypticase soy agar ( TSA ) to obtain viable counts. Since heat treatment kills the vegetative

  8. Differences in Susceptibility of Inbred Mice to Bacillus anthracis

    Science.gov (United States)

    1985-04-26

    dilutions of the mixture were prepared and injected into A/J and CBA/J mice via the tail vein, as described by Ezzell et al. (9). Five mice per strain were...xylazine (Rompun, Miles Laboratories, Shawnee, Kansas) in 50 pl, and were dissected iwnmediately. Gross pathological changes were noted, heart blood and...anthracis; a histopathological study of skin lesions produced by B. anthracis in susceptible and resistant animal species. J. Infect. Dis. 80:1-13. 9. Ezzell

  9. Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides.

    Science.gov (United States)

    Kobayashi, Kaori; Guilliam, Thomas A; Tsuda, Masataka; Yamamoto, Junpei; Bailey, Laura J; Iwai, Shigenori; Takeda, Shunichi; Doherty, Aidan J; Hirota, Kouji

    2016-08-02

    PrimPol is a DNA damage tolerance enzyme possessing both translesion synthesis (TLS) and primase activities. To uncover its potential role in TLS-mediated IgVλ hypermutation and define its interplay with other TLS polymerases, PrimPol(-/-) and PrimPol(-/-)/Polη(-/-)/Polζ (-/-) gene knockouts were generated in avian cells. Loss of PrimPol had no significant impact on the rate of hypermutation or the mutation spectrum of IgVλ. However, PrimPol(-/-) cells were sensitive to methylmethane sulfonate, suggesting that it may bypass abasic sites at the IgVλ segment by repriming DNA synthesis downstream of these sites. PrimPol(-/-) cells were also sensitive to cisplatin and hydroxyurea, indicating that it assists in maintaining / restarting replication at a variety of lesions. To accurately measure the relative contribution of the TLS and primase activities, we examined DNA damage sensitivity in PrimPol(-/-) cells complemented with polymerase or primase-deficient PrimPol. Polymerase-defective, but not primase-deficient, PrimPol suppresses the hypersensitivity of PrimPol(-/-) cells. This indicates that its primase, rather than TLS activity, is pivotal for DNA damage tolerance. Loss of TLS polymerases, Polη and Polζ has an additive effect on the sensitivity of PrimPol(-/-) cells. Moreover, we found that PrimPol and Polη-Polζ redundantly prevented cell death and facilitated unperturbed cell cycle progression. PrimPol(-/-) cells also exhibited increased sensitivity to a wide variety of chain-terminating nucleoside analogs (CTNAs). PrimPol could perform close-coupled repriming downstream of CTNAs and oxidative damage in vitro. Together, these results indicate that PrimPol's repriming activity plays a central role in reinitiating replication downstream from CTNAs and other specific DNA lesions.

  10. Rapid-viability PCR method for detection of live, virulent Bacillus anthracis in environmental samples.

    Science.gov (United States)

    Létant, Sonia E; Murphy, Gloria A; Alfaro, Teneile M; Avila, Julie R; Kane, Staci R; Raber, Ellen; Bunt, Thomas M; Shah, Sanjiv R

    2011-09-01

    In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.

  11. Novel giant siphovirus from Bacillus anthracis features unusual genome characteristics.

    Directory of Open Access Journals (Sweden)

    Holly H Ganz

    Full Text Available Here we present vB_BanS-Tsamsa, a novel temperate phage isolated from Bacillus anthracis, the agent responsible for anthrax infections in wildlife, livestock and humans. Tsamsa phage is a giant siphovirus (order Caudovirales, featuring a long, flexible and non-contractile tail of 440 nm (not including baseplate structure and an isometric head of 82 nm in diameter. We induced Tsamsa phage in samples from two different carcass sites in Etosha National Park, Namibia. The Tsamsa phage genome is the largest sequenced Bacillus siphovirus, containing 168,876 bp and 272 ORFs. The genome features an integrase/recombinase enzyme, indicative of a temperate lifestyle. Among bacterial strains tested, the phage infected only certain members of the Bacillus cereus sensu lato group (B. anthracis, B. cereus and B. thuringiensis and exhibited moderate specificity for B. anthracis. Tsamsa lysed seven out of 25 B. cereus strains, two out of five B. thuringiensis strains and six out of seven B. anthracis strains tested. It did not lyse B. anthracis PAK-1, an atypical strain that is also resistant to both gamma phage and cherry phage. The Tsamsa endolysin features a broader lytic spectrum than the phage host range, indicating possible use of the enzyme in Bacillus biocontrol.

  12. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  13. Methods of introducing nucleic acids into cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Lajoie, Marc J.; Gregg, Christopher J.; Mosberg, Joshua A.; Church, George M.

    2017-06-27

    A method of introducing a nucleic acid sequence into a cell is provided where the cell has impaired or inhibited or disrupted DnaG primase activity or impaired or inhibited or disrupted DnaB helicase activity, or larger or increased gaps or distance between Okazaki fragments or lowered or reduced frequency of Okazaki fragment initiation, or the cell has increased single stranded DNA (ssDNA) on the lagging strand of the replication fork including transforming the cell through recombination with a nucleic acid oligomer.

  14. Computational Fluid Dynamics Modeling of Bacillus anthracis ...

    Science.gov (United States)

    Journal Article Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived from computed tomography (CT) or µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation-exhalation breathing conditions using average species-specific minute volumes. Four different exposure scenarios were modeled in the rabbit based upon experimental inhalation studies. For comparison, human simulations were conducted at the highest exposure concentration used during the rabbit experimental exposures. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Despite the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the upper conducting airways of the human at the same air concentration of anthrax spores. This greater deposition of spores in the upper airways in the human resulted in lower penetration and deposition in the tracheobronchial airways and the deep lung than that predict

  15. Alleged B. anthracis exposure claims in a workers' compensation setting.

    Science.gov (United States)

    Jewell, Gregory; Dunning, Kari; Lockey, James E

    2006-01-01

    Workers' compensation insurance in some states may not provide coverage for medical evaluation costs of workplace exposures related to potential bioterrorism acts if there is no diagnosed illness or disease. Personal insurance also may not provide coverage for these exposures occurring at the workplace. Governmental entities, insurers, and employers need to consider how to address such situations and the associated costs. The objective of this study was to examine characteristics of workers and total costs associated with workers' compensation claims alleging potential exposure to the bioterrorism organism B. anthracis. We examined 192 claims referred for review to the Ohio Bureau of Workers' Compensation (OBWC) from October 10, 2001, through December 20, 2004. Although some cases came from out-of-state areas where B. anthracis exposure was known to exist, no Ohio claim was associated with true B. anthracis exposure or B. anthracis-related illness. Of the 155 eligible claims, 126 included medical costs averaging dollar 219 and ranging from dollar 24 to dollar 3,126. There was no difference in mean cost for government and non-government employees (p = 0.202 Wilcoxon). The number of claims and associated medical costs for evaluation and treatment of potential workplace exposure to B. anthracis were relatively small. These results can be attributed to several factors, including no documented B. anthracis exposures and disease in Ohio and prompt transmission of recommended diagnostic and prophylactic treatment protocols to physicians. How employers, insurers, and jurisdictions address payment for evaluation and treatment of potential or documented exposures resulting from a potential terrorism-related event should be addressed proactively.

  16. [Clustered regularly interspaced short palindromic repeats (CRISPR) site in Bacillus anthracis].

    Science.gov (United States)

    Gao, Zhiqi; Wang, Dongshu; Feng, Erling; Wang, Bingxiang; Hui, Yiming; Han, Shaobo; Jiao, Lei; Liu, Xiankai; Wang, Hengliang

    2014-11-04

    To investigate the polymorphism of clustered regularly interspaced short palindromic repeats (CRISPR) in Bacillu santhracis and the application to molecular typing based on the polymorphism of CRISPR in B. anthracis. We downloaded the whole genome sequence of 6 B. anthracis strains and extracted the CRISPR sites. We designed the primers of CRISPR sites and amplified the CRISPR fragments in 193 B. anthracis strains by PCR and sequenced these fragments. In order to reveal the polymorphism of CRISPR in B. anthracis, wealigned all the extracted sequences and sequenced results by local blasting. At the same time, we also analyzed the CRISPR sites in B. cereus and B. thuringiensis. We did not find any polymorphism of CRISPR in B. anthracis. The molecular typing approach based on CRISPR polymorphism is not suitable for B. anthracis, but it is possible for us to distinguish B. anthracis from B. cereus and B. thuringiensis.

  17. Preliminary report for analysis of genome wide mutations from four ciprofloxacin resistant B. anthracis Sterne isolates generated by Illumina, 454 sequencing and microarrays for DHS

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, Crystal [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vergez, Lisa [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Hinckley, Aubree [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Thissen, James [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Gardner, Shea [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); McLoughlin, Kevin [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Jackson, Paul [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Ellingson, Sally [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Hauser, Loren [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Brettin, Tom [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Fofanov, Viacheslav [Eureka Genomics, Hercules, CA (United States); Koshinsky, Heather [Eureka Genomics, Hercules, CA (United States); Fofanov, Yuriy [Univ. of Houston, TX (United States)

    2011-06-21

    The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, Taqman PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. As the result of a different DHS project, we have selected for and isolated a large number of ciprofloxacin resistant B. anthracis Sterne isolates. These isolates vary in the concentrations of ciprofloxacin that they can tolerate, suggesting multiple mutations in the samples. In collaboration with University of Houston, Eureka Genomics and Oak Ridge National Laboratory, we analyzed the ciprofloxacin resistant B. anthracis Sterne isolates by microarray hybridization, Illumina and Roche 454 sequencing to understand the error rates and sensitivity of the different methods. The report provides an assessment of the results and a complete set of all protocols used and all data generated along with information to interpret the protocols and data sets.

  18. Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, P.J.; Walthers, E.A.; Richmond, K.L. [Los Alamos National Lab., NM (United States)] [and others

    1997-04-01

    PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five Polymorphisms differed by the presence Of two to six copies of the 12-bp tandem repeat 5{prime}-CAATATCAACAA-3{prime}. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats are generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations. 22 refs., 4 figs., 3 tabs.

  19. DECONTAMINATION ASSESSMENT OF BACILLUS ANTHRACIS, BACILLUS SUBTILIS, AND GEOBACILLUS STEAROTHERMOPHILUS SPORES ON INDOOR SURFACTS USING A HYDROGEN PERIOXIDE GAS GENERATOR

    Science.gov (United States)

    Aims: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. Methods and Results: B. anthracis, B. subtilis, and G. Stearothermophilus spores were dried on seven...

  20. Method for screening inhibitors of the toxicity of Bacillus anthracis

    Science.gov (United States)

    Cirino, Nick M.; Jackson, Paul J.; Lehnert, Bruce E.

    2001-01-01

    The protective antigen (PA) of Bacillus anthracis is integral to the mechanism of anthrax poisoning. The cloning, expression and purification of a 32 kDa B. anthracis PA fragment (PA32) is described. This fragment has also been expressed as a fusion construct to stabilized green fluorescent protein (EGFP-PA32). Both proteins were capable of binding to specific cell surface receptors as determined by fluorescent microscopy and a flow cytometric assay. To confirm binding specificity in the flow cytometric assay, non-fluorescent PA83 or PA32 was used to competitively inhibit fluorescent EGFP-PA32 binding to cell receptors. This assay can be employed as a rapid screen for compounds which disrupts binding of PA to cells. Additionally, the high intracellular expression levels and ease of purification make this recombinant protein an attractive vaccine candidate or therapeutic treatment for anthrax poisoning.

  1. Historical distribution and molecular diversity of Bacillus anthracis, Kazakhstan.

    Science.gov (United States)

    Aikembayev, Alim M; Lukhnova, Larissa; Temiraliyeva, Gulnara; Meka-Mechenko, Tatyana; Pazylov, Yerlan; Zakaryan, Sarkis; Denissov, Georgiy; Easterday, W Ryan; Van Ert, Matthew N; Keim, Paul; Francesconi, Stephen C; Blackburn, Jason K; Hugh-Jones, Martin; Hadfield, Ted

    2010-05-01

    To map the distribution of anthrax outbreaks and strain subtypes in Kazakhstan during 1937-2005, we combined geographic information system technology and genetic analysis by using archived cultures and data. Biochemical and genetic tests confirmed the identity of 93 archived cultures in the Kazakhstan National Culture Collection as Bacillus anthracis. Multilocus variable number tandem repeat analysis genotyping identified 12 genotypes. Cluster analysis comparing these genotypes with previously published genotypes indicated that most (n = 78) isolates belonged to the previously described A1.a genetic cluster, 6 isolates belonged to the A3.b cluster, and 2 belonged to the A4 cluster. Two genotypes in the collection appeared to represent novel genetic sublineages; 1 of these isolates was from Krygystan. Our data provide a description of the historical, geographic, and genetic diversity of B. anthracis in this Central Asian region.

  2. Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh.

    Science.gov (United States)

    Rume, Farzana Islam; Affuso, Alessia; Serrecchia, Luigina; Rondinone, Valeria; Manzulli, Viviana; Campese, Emanuele; Di Taranto, Pietro; Biswas, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Yasmin, Mahmuda; Fasanella, Antonio; Hugh-Jones, Martin

    2016-01-01

    In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.

  3. Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh.

    Directory of Open Access Journals (Sweden)

    Farzana Islam Rume

    Full Text Available In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA with the analysis of 15 Variable Number Tandem Repeats (VNTR, demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.

  4. Nanoscale Structural and Mechanical Analysis of Bacillus anthracis Spores Inactivated with Rapid Dry Heating

    Science.gov (United States)

    Felker, Daniel L.; Burggraf, Larry W.

    2014-01-01

    Effective killing of Bacillus anthracis spores is of paramount importance to antibioterrorism, food safety, environmental protection, and the medical device industry. Thus, a deeper understanding of the mechanisms of spore resistance and inactivation is highly desired for developing new strategies or improving the known methods for spore destruction. Previous studies have shown that spore inactivation mechanisms differ considerably depending upon the killing agents, such as heat (wet heat, dry heat), UV, ionizing radiation, and chemicals. It is believed that wet heat kills spores by inactivating critical enzymes, while dry heat kills spores by damaging their DNA. Many studies have focused on the biochemical aspects of spore inactivation by dry heat; few have investigated structural damages and changes in spore mechanical properties. In this study, we have inactivated Bacillus anthracis spores with rapid dry heating and performed nanoscale topographical and mechanical analysis of inactivated spores using atomic force microscopy (AFM). Our results revealed significant changes in spore morphology and nanomechanical properties after heat inactivation. In addition, we also found that these changes were different under different heating conditions that produced similar inactivation probabilities (high temperature for short exposure time versus low temperature for long exposure time). We attributed the differences to the differential thermal and mechanical stresses in the spore. The buildup of internal thermal and mechanical stresses may become prominent only in ultrafast, high-temperature heat inactivation when the experimental timescale is too short for heat-generated vapor to efficiently escape from the spore. Our results thus provide direct, visual evidences of the importance of thermal stresses and heat and mass transfer to spore inactivation by very rapid dry heating. PMID:24375142

  5. Rapid detection and identification of Bacillus anthracis in food using pyrosequencing technology.

    Science.gov (United States)

    Amoako, Kingsley K; Janzen, Timothy W; Shields, Michael J; Hahn, Kristen R; Thomas, Matthew C; Goji, Noriko

    2013-08-01

    The development of advanced methodologies for the detection of Bacillus anthracis has been evolving rapidly since the release of the anthrax spores in the mail in 2001. Recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence based such as pyrosequencing, which has the capability to determine short DNA stretches in real-time using biotinylated PCR amplicons, has potential biodefense applications. Using markers from the virulence plasmids (pXO1 and pXO2) and chromosomal regions, we have demonstrated the power of this technology in the rapid, specific and sensitive detection of B. anthracis spores in food matrices including milk, juice, bottled water, and processed meat. The combined use of immunomagnetic separation and pyrosequencing showed positive detection when liquid foods (bottled water, milk, juice), and processed meat were experimentally inoculated with 6CFU/mL and 6CFU/g, respectively, without an enrichment step. Pyrosequencing is completed in about 60min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence. The entire assay (from sample preparation to sequencing information) can be completed in about 7.5h. A typical run on food samples yielded 67-80bp reads with 94-100% identity to the expected sequence. This sequence based approach is a novel application for the detection of anthrax spores in food with potential application in foodborne bioterrorism response and biodefense involving the use of anthrax spores. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  6. Encapsulated Bacillus anthracis interacts closely with liver endothelium.

    Science.gov (United States)

    Piris-Gimenez, Alejandro; Corre, Jean-Philippe; Jouvion, Gregory; Candela, Thomas; Khun, Huot; Goossens, Pierre L

    2009-11-01

    The Bacillus anthracis poly-gamma-D-glutamate capsule is essential for virulence. It impedes phagocytosis and protects bacilli from the immune system, thus promoting systemic dissemination. To further define the virulence mechanisms brought into play by the capsule, we characterized the interactions between encapsulated nontoxinogenic B. anthracis and its host in vivo through histological analysis, perfusion, and competition experiments with purified capsule. Clearance of encapsulated bacilli from the blood was rapid (>90% clearance within 5 min), with 75% of the bacteria being trapped in the liver. Competition experiments with purified capsule polyglutamate inhibited this interaction. At the septicemic phase of cutaneous infection with spores, the encapsulated bacilli were trapped in the vascular spaces of the liver and interacted closely with the liver endothelium in the sinusoids and terminal and portal veins. They often grow as microcolonies containing capsular material shed by the bacteria. We show that, in addition to its inhibitory effect on the interaction with the immune system, the capsule surrounding B. anthracis plays an active role in mediating the trapping of the bacteria within the liver and may thus contribute to anthrax pathogenesis. Because other microorganisms produce polyglutamate, it may also represent a general mechanism of virulence or in vivo survival.

  7. Toxin-independent virulence of Bacillus anthracis in rabbits.

    Directory of Open Access Journals (Sweden)

    Haim Levy

    Full Text Available The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play a major role in pathogenicity. In the guinea pig (GP model we have previously shown that deletion of all three toxin components results in a relatively moderate attenuation in virulence, indicating that B. anthracis possesses an additional toxin-independent virulence mechanism. To characterize this toxin-independent mechanism in anthrax disease, we developed a new rabbit model by intravenous injection (IV of B. anthracis encapsulated vegetative cells, artificially creating bacteremia. Using this model we were able to demonstrate that also in rabbits, B. anthracis mutants lacking the toxins are capable of killing the host within 24 hours. This virulent trait depends on the activity of AtxA in the presence of pXO2, as, in the absence of the toxin genes, deletion of either component abolishes virulence. Furthermore, this IV virulence depends mainly on AtxA rather than the whole pXO1. A similar pattern was shown in the GP model using subcutaneous (SC administration of spores of the mutant strains, demonstrating the generality of the phenomenon. The virulent strains showed higher bacteremia levels and more efficient tissue dissemination; however our interpretation is that tissue dissemination per se is not the main determinant of virulence whose exact nature requires further elucidation.

  8. Survival of Bacillus anthracis spores in fruit juices and wine.

    Science.gov (United States)

    Leishman, Oriana N; Johnson, Miranda J; Labuza, Theodore P; Diez-Gonzalez, Francisco

    2010-09-01

    Foods have been identified as a potential target for bioterrorism due to their essential nature and global distribution. Foods produced in bulk have the potential to have large batches of product intentionally contaminated, which could affect hundreds or thousands of individuals. Bacillus anthracis spores are one potential bioterrorism agent that may survive pasteurization and remain viable throughout the shelf life of fruit juices and cause disease if consumed. This project examined B. anthracis spore survival in orange, apple, and grape juices, as well as wine. Samples of beverages were inoculated with spores of two nonpathogenic B. anthracis strains at approximately 10(6) CFU/ml, and the spore count was determined periodically during storage for 30 days at 4°C. After this time, the counts of survival spores never declined more than 1 log CFU/ml in any of the beverage types. These results indicate that spores can survive, with little to no loss in viability, for at least a month in fruit juices and wine.

  9. Assembly and Function of the Bacillus anthracis S-Layer.

    Science.gov (United States)

    Missiakas, Dominique; Schneewind, Olaf

    2017-09-08

    Bacillus anthracis, the anthrax agent, is a member of the Bacillus cereus sensu lato group, which includes invasive pathogens of mammals or insects as well as nonpathogenic environmental strains. The genes for anthrax pathogenesis are located on two large virulence plasmids. Similar virulence plasmids have been acquired by other B. cereus strains and enable the pathogenesis of anthrax-like diseases. Among the virulence factors of B. anthracis is the S-layer-associated protein BslA, which endows bacilli with invasive attributes for mammalian hosts. BslA surface display and function are dependent on the bacterial S-layer, whose constituents assemble by binding to the secondary cell wall polysaccharide (SCWP) via S-layer homology (SLH) domains. B. anthracis and other pathogenic B. cereus isolates harbor genes for the secretion of S-layer proteins, for S-layer assembly, and for synthesis of the SCWP. We review here recent insights into the assembly and function of the S-layer and the SCWP.

  10. Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

    Directory of Open Access Journals (Sweden)

    Ciervo Alessandra

    2006-04-01

    Full Text Available Abstract Background The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR Analysis (MLVA assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. Results Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. Conclusion In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the

  11. In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication.

    Science.gov (United States)

    Schauer, Grant; Finkelstein, Jeff; O'Donnell, Mike

    2017-09-20

    The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their 'correct' strands, as well as quality control mechanisms that evict polymerases when they associate with an 'incorrect' strand. Here, we provide a practical guide to differentially assay leading and lagging strand replication in vitro using pure proteins.

  12. Hal Is a Bacillus anthracis Heme Acquisition Protein

    Science.gov (United States)

    Balderas, Miriam A.; Nobles, Christopher L.; Honsa, Erin S.; Alicki, Embriette R.

    2012-01-01

    The metal iron is a limiting nutrient for bacteria during infection. Bacillus anthracis, the causative agent of anthrax and a potential weapon of bioterrorism, grows rapidly in mammalian hosts, which suggests that it efficiently attains iron during infection. Recent studies have uncovered both heme (isd) and siderophore-mediated (asb) iron transport pathways in this pathogen. Whereas deletion of the asb genes results in reduced virulence, the loss of three surface components from isd had no effect, thereby leaving open the question of what additional factors in B. anthracis are responsible for iron uptake from the most abundant iron source for mammals, heme. Here, we describe the first functional characterization of bas0520, a gene recently implicated in anthrax disease progression. bas0520 encodes a single near-iron transporter (NEAT) domain and several leucine-rich repeats. The NEAT domain binds heme, despite lacking a stabilizing tyrosine common to the NEAT superfamily of hemoproteins. The NEAT domain also binds hemoglobin and can acquire heme from hemoglobin in solution. Finally, deletion of bas0520 resulted in bacilli unable to grow efficiently on heme or hemoglobin as an iron source and yielded the most significant phenotype relative to that for other putative heme uptake systems, a result that suggests that this protein plays a prominent role in the replication of B. anthracis in hematogenous environments. Thus, we have assigned the name of Hal (heme-acquisition leucine-rich repeat protein) to BAS0520. These studies advance our understanding of heme acquisition by this dangerous pathogen and justify efforts to determine the mechanistic function of this novel protein for vaccine or inhibitor development. PMID:22865843

  13. [Survival of Bacillus anthracis spores in various tannery baths].

    Science.gov (United States)

    Mendrycka, M; Mierzejewski, J

    2000-01-01

    The influence of tannery baths: liming, deliming, bating, pickling, tanning, retannage on the survival and on the germination dynamism of B. anthracis spores (Sterne strain) was investigated. The periods and the conditions of this influence were established according to technological process of cow hide tannage. Practically after every bath some part of the spores remained vital. The most effective killing of spores occurred after pickling, liming and deliming. Inversely, the most viable spores remained after bating and retannage process. The lack of correlation that was observed between survival and germination of spores after retannage bath can be explained by different mechanism of spores germination inhibition and their killing.

  14. Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication

    Science.gov (United States)

    Seco, Elena M.

    2017-01-01

    Abstract Firmicutes have two distinct replicative DNA polymerases, the PolC leading strand polymerase, and PolC and DnaE synthesizing the lagging strand. We have reconstituted in vitro Bacillus subtilis bacteriophage SPP1 θ-type DNA replication, which initiates unidirectionally at oriL. With this system we show that DnaE is not only restricted to lagging strand synthesis as previously suggested. DnaG primase and DnaE polymerase are required for initiation of DNA replication on both strands. DnaE and DnaG synthesize in concert a hybrid RNA/DNA ‘initiation primer’ on both leading and lagging strands at the SPP1 oriL region, as it does the eukaryotic Pol α complex. DnaE, as a RNA-primed DNA polymerase, extends this initial primer in a reaction modulated by DnaG and one single-strand binding protein (SSB, SsbA or G36P), and hands off the initiation primer to PolC, a DNA-primed DNA polymerase. Then, PolC, stimulated by DnaG and the SSBs, performs the bulk of DNA chain elongation at both leading and lagging strands. Overall, these modulations by the SSBs and DnaG may contribute to the mechanism of polymerase switch at Firmicutes replisomes. PMID:28575448

  15. Characterization of the sortase repertoire in Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Willy Aucher

    Full Text Available LPXTG proteins, present in most if not all Gram-positive bacteria, are known to be anchored by sortases to the bacterial peptidoglycan. More than one sortase gene is often encoded in a bacterial species, and each sortase is supposed to specifically anchor given LPXTG proteins, depending of the sequence of the C-terminal cell wall sorting signal (cwss, bearing an LPXTG motif or another recognition sequence. B. anthracis possesses three sortase genes. B. anthracis sortase deleted mutant strains are not affected in their virulence. To determine the sortase repertoires, we developed a genetic screen using the property of the gamma phage to lyse bacteria only when its receptor, GamR, an LPXTG protein, is exposed at the surface. We identified 10 proteins that contain a cell wall sorting signal and are covalently anchored to the peptidoglycan. Some chimeric proteins yielded phage lysis in all sortase mutant strains, suggesting that cwss proteins remained surface accessible in absence of their anchoring sortase, probably as a consequence of membrane localization of yet uncleaved precursor proteins. For definite assignment of the sortase repertoires, we consequently relied on a complementary test, using a biochemical approach, namely immunoblot experiments. The sortase anchoring nine of these proteins has thus been determined. The absence of virulence defect of the sortase mutants could be a consequence of the membrane localization of the cwss proteins.

  16. Bacillus anthracis genome organization in light of whole transcriptome sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Jeffrey; Zhu, Wenhan; Passalacqua, Karla D.; Bergman, Nicholas; Borodovsky, Mark

    2010-03-22

    Emerging knowledge of whole prokaryotic transcriptomes could validate a number of theoretical concepts introduced in the early days of genomics. What are the rules connecting gene expression levels with sequence determinants such as quantitative scores of promoters and terminators? Are translation efficiency measures, e.g. codon adaptation index and RBS score related to gene expression? We used the whole transcriptome shotgun sequencing of a bacterial pathogen Bacillus anthracis to assess correlation of gene expression level with promoter, terminator and RBS scores, codon adaptation index, as well as with a new measure of gene translational efficiency, average translation speed. We compared computational predictions of operon topologies with the transcript borders inferred from RNA-Seq reads. Transcriptome mapping may also improve existing gene annotation. Upon assessment of accuracy of current annotation of protein-coding genes in the B. anthracis genome we have shown that the transcriptome data indicate existence of more than a hundred genes missing in the annotation though predicted by an ab initio gene finder. Interestingly, we observed that many pseudogenes possess not only a sequence with detectable coding potential but also promoters that maintain transcriptional activity.

  17. Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity.

    Science.gov (United States)

    Hammerstrom, Troy G; Horton, Lori B; Swick, Michelle C; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M

    2015-02-01

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO(2)/bicarbonate, and there is a positive correlation between the CO(2)/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His→Asp) and phosphoablative (His→Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism. © 2014 John Wiley & Sons Ltd.

  18. In Silico Genomic Fingerprints of the Bacillus anthracis Group Obtained by Virtual Hybridization

    Directory of Open Access Journals (Sweden)

    Hueman Jaimes-Díaz

    2015-02-01

    Full Text Available In this study we evaluate the capacity of Virtual Hybridization to identify between highly related bacterial strains. Eight genomic fingerprints were obtained by virtual hybridization for the Bacillus anthracis genome set, and a set of 15,264 13-nucleotide short probes designed to produce genomic fingerprints unique for each organism. The data obtained from each genomic fingerprint were used to obtain hybridization patterns simulating a DNA microarray. Two virtual hybridization methods were used: the Direct and the Extended method to identify the number of potential hybridization sites and thus determine the minimum sensitivity value to discriminate between genomes with 99.9% similarity. Genomic fingerprints were compared using both methods and phylogenomic trees were constructed to verify that the minimum detection value is 0.000017. Results obtained from the genomic fingerprints suggest that the distribution in the trees is correct, as compared to other taxonomic methods. Specific virtual hybridization sites for each of the genomes studied were also identified.

  19. FORMALDEHYDE GAS INACTIVATION OF BACILLUS ANTHRACIS, BACILLUS SUBTILIS AND GEOBACILLUS STEAROTHERMOPHILUS SPORES ON INDOOR SURFACE MATERIALS.

    Science.gov (United States)

    Research evaluated the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface material using formaldehyde gas. Spores were dried on seven types of indoor surfaces and exposed to 1100 ppm formaldehyde gas for 10 hr. Fo...

  20. Efficacy of Oritavancin in a Murine Model of Bacillus anthracis Spore Inhalation Anthrax

    National Research Council Canada - National Science Library

    Heine, H. S; Bassett, J; Miller, L; Bassett, A; Ivins, B. E; Lehous, D; Arhin, F. F; Parr, Jr., T. R; Moeck, G

    2008-01-01

    The inhaled form of Bacillus anthracis infection may be fatal to humans. The current standard of care for inhalational anthrax postexposure prophylaxis is ciprofloxacin therapy twice daily for 60 days...

  1. Lethality of Bacillus Anthracis Spores Due to Short Duration Heating Measured Using Infrared Spectroscopy

    National Research Council Canada - National Science Library

    Goetz, Kristina M

    2005-01-01

    In this research, Bacillus anthracis spores were subjected to bursts of heat lasting on the order of one second in duration using a laser system to simulate the explosive environment from an agent defeat weapon...

  2. Ecological Niche Modelling of the Bacillus anthracis A1.a sub-lineage in Kazakhstan

    Science.gov (United States)

    2011-01-01

    Background Bacillus anthracis, the causative agent of anthrax, is a globally distributed zoonotic pathogen that continues to be a veterinary and human health problem in Central Asia. We used a database of anthrax outbreak locations in Kazakhstan and a subset of genotyped isolates to model the geographic distribution and ecological associations of B. anthracis in Kazakhstan. The aims of the study were to test the influence of soil variables on a previous ecological niche based prediction of B. anthracis in Kazakhstan and to determine if a single sub-lineage of B. anthracis occupies a unique ecological niche. Results The addition of soil variables to the previously developed ecological niche model did not appreciably alter the limits of the predicted geographic or ecological distribution of B. anthracis in Kazakhstan. The A1.a experiment predicted the sub-lineage to be present over a larger geographic area than did the outbreak based experiment containing multiple lineages. Within the geographic area predicted to be suitable for B. anthracis by all ten best subset models, the A1.a sub-lineage was associated with a wider range of ecological tolerances than the outbreak-soil experiment. Analysis of rule types showed that logit rules predominate in the outbreak-soil experiment and range rules in the A1.a sub-lineage experiment. Random sub-setting of locality points suggests that models of B. anthracis distribution may be sensitive to sample size. Conclusions Our analysis supports careful consideration of the taxonomic resolution of data used to create ecological niche models. Further investigations into the environmental affinities of individual lineages and sub-lineages of B. anthracis will be useful in understanding the ecology of the disease at large and small scales. With model based predictions serving as approximations of disease risk, these efforts will improve the efficacy of public health interventions for anthrax prevention and control. PMID:22152056

  3. Functional and Immunological Analyses of Superoxide Dismutases and Other Spore-Associated Proteins of Bacillus anthracis

    Science.gov (United States)

    2008-08-20

    L., S. Hibbs, P. Tsai, G. L. Cao, and G. M. Rosen . 2005. Role of superoxide in the germination of Bacillus anthracis endospores. FEMS Microbiol...178:7994-8001. 42. Cohen, S., I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak ...K. W. Raines, G. L. Cao, S. Hibbs, P. Tsai, L. Baillie, G. M. Rosen , and A. S. Cross. 2007. Protective role of Bacillus anthracis exosporium in

  4. Identification of Bacillus anthracis by Using Monoclonal Antibody to Cell Wall Galactose-N-Acetylglucosamine Polysaccharide

    Science.gov (United States)

    1990-02-01

    Bacillus circulans ATCC 4513 b - - NR NT NT NT NT Bacillus coagulans ATCC 7050 b - - NR NT NT NT NT Bacillus eugilitis B-61 f - - NR NT NT NT NT...American Society for Microbiology W Identification of Bacillus anthracis by-U-sing Monoclonal Antibody CC to Cell Wall Galactose-N-Acetylglucosamine...Received 22 June 1989/Accepted 31 October 1989 ’ Guanidine extracts of crude Bacillus anthracis cell wall were used to vaccinate BALB/c mice and to

  5. The secret life of the anthrax agent Bacillus anthracis: bacteriophage-mediated ecological adaptations.

    Directory of Open Access Journals (Sweden)

    Raymond Schuch

    2009-08-01

    Full Text Available Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities.

  6. Caenorhabditis elegans Predation on Bacillus anthracis: Decontamination of Spore Contaminated Soil with Germinants and Nematodes.

    Science.gov (United States)

    Schelkle, Bettina; Choi, Young; Baillie, Leslie W; Richter, William; Buyuk, Fatih; Celik, Elif; Wendling, Morgan; Sahin, Mitat; Gallagher, Theresa

    2017-01-01

    Remediation of Bacillus anthracis -contaminated soil is challenging and approaches to reduce overall spore levels in environmentally contaminated soil or after intentional release of the infectious disease agent in a safe, low-cost manner are needed. B. anthracis spores are highly resistant to biocides, but once germinated they become susceptible to traditional biocides or potentially even natural predators such as nematodes in the soil environment. Here, we describe a two-step approach to reducing B. anthracis spore load in soil during laboratory trials, whereby germinants and Caenorhabditis elegans nematodes are applied concurrently. While the application of germinants reduced B. anthracis spore load by up to four logs depending on soil type, the addition of nematodes achieved a further log reduction in spore count. These laboratory based results suggest that the combined use of nematodes and germinants could represent a promising approach for the remediation of B. anthracis spore contaminated soil. Originality-Significance Statement: This study demonstrates for the first time the successful use of environmentally friendly decontamination methods to inactivate Bacillus anthracis spores in soil using natural predators of the bacterium, nematode worms.

  7. Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates.

    Science.gov (United States)

    Stengel, Gudrun; Kuchta, Robert D

    2011-01-01

    The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not

  8. Decontamination Options for Drinking Water Contaminated with Bacillus anthracis Spores

    Energy Technology Data Exchange (ETDEWEB)

    Raber, E; Burklund, A

    2010-02-16

    Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination options for use in a contaminated drinking water supply. The parameters were: (1) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus); (2) spore concentration in suspension (10{sup 2} to 10{sup 6} spores/ml); (3) chemical characteristics of decontaminant [sodium dicholor-s-triazinetrione dihydrate (Dichlor), hydrogen peroxide, potassium peroxymonosulfate (Oxone), sodium hypochlorite, and VirkonS{reg_sign}]; (4) decontaminant concentration (0.01% to 5%); and (5) decontaminant exposure time (10 min to 24 hr). Results from 162 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5%, and Dichlor and sodium hypochlorite at a concentration of 2%, were effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor's desirable characteristics of high oxidation potential, high level of free chlorine, and more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting EPA's biocide standard of greater than a 6 log kill after a 10-minute exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS{reg_sign} and Oxone were less effective decontaminants than other options evaluated in this study and did not meet the EPA's efficacy standard for biocides. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult.

  9. Distribution and molecular evolution of bacillus anthracis genotypes in Namibia.

    Directory of Open Access Journals (Sweden)

    Wolfgang Beyer

    Full Text Available The recent development of genetic markers for Bacillus anthracis has made it possible to monitor the spread and distribution of this pathogen during and between anthrax outbreaks. In Namibia, anthrax outbreaks occur annually in the Etosha National Park (ENP and on private game and livestock farms. We genotyped 384 B. anthracis isolates collected between 1983-2010 to identify the possible epidemiological correlations of anthrax outbreaks within and outside the ENP and to analyze genetic relationships between isolates from domestic and wild animals. The isolates came from 20 animal species and from the environment and were genotyped using a 31-marker multi-locus-VNTR-analysis (MLVA and, in part, by twelve single nucleotide polymorphism (SNP markers and four single nucleotide repeat (SNR markers. A total of 37 genotypes (GT were identified by MLVA, belonging to four SNP-groups. All GTs belonged to the A-branch in the cluster- and SNP-analyses. Thirteen GTs were found only outside the ENP, 18 only within the ENP and 6 both inside and outside. Genetic distances between isolates increased with increasing time between isolations. However, genetic distance between isolates at the beginning and end of the study period was relatively small, indicating that while the majority of GTs were only found sporadically, three genetically close GTs, accounting for more than four fifths of all the ENP isolates, appeared dominant throughout the study period. Genetic distances among isolates were significantly greater for isolates from different host species, but this effect was small, suggesting that while species-specific ecological factors may affect exposure processes, transmission cycles in different host species are still highly interrelated. The MLVA data were further used to establish a model of the probable evolution of GTs within the endemic region of the ENP. SNR-analysis was helpful in correlating an isolate with its source but did not elucidate

  10. Bacillus anthracis in China and its relationship to worldwide lineages

    Directory of Open Access Journals (Sweden)

    Schupp James M

    2009-04-01

    Full Text Available Abstract Background The global pattern of distribution of 1033 B. anthracis isolates has previously been defined by a set of 12 conserved canonical single nucleotide polymorphisms (canSNP. These studies reinforced the presence of three major lineages and 12 sub-lineages and sub-groups of this anthrax-causing pathogen. Isolates that form the A lineage (unlike the B and C lineages have become widely dispersed throughout the world and form the basis for the geographical disposition of "modern" anthrax. An archival collection of 191 different B. anthracis isolates from China provides a glimpse into the possible role of Chinese trade and commerce in the spread of certain sub-lineages of this pathogen. Canonical single nucleotide polymorphism (canSNP and multiple locus VNTR analysis (MLVA typing has been used to examine this archival collection of isolates. Results The canSNP study indicates that there are 5 different sub-lineages/sub-groups in China out of 12 previously described world-wide canSNP genotypes. Three of these canSNP genotypes were only found in the western-most province of China, Xinjiang. These genotypes were A.Br.008/009, a sub-group that is spread across most of Europe and Asia; A.Br.Aust 94, a sub-lineage that is present in Europe and India, and A.Br.Vollum, a lineage that is also present in Europe. The remaining two canSNP genotypes are spread across the whole of China and belong to sub-group A.Br.001/002 and the A.Br.Ames sub-lineage, two closely related genotypes. MLVA typing adds resolution to the isolates in each canSNP genotype and diversity indices for the A.Br.008/009 and A.Br.001/002 sub-groups suggest that these represent older and established clades in China. Conclusion B. anthracis isolates were recovered from three canSNP sub-groups (A.Br.008/009, A.Br.Aust94, and A.Br.Vollum in the western most portion of the large Chinese province of Xinjiang. The city of Kashi in this province appears to have served as a crossroads

  11. Decontamination Efficacy and Skin Toxicity of Two Decontaminants against Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Chad W Stratilo

    Full Text Available Decontamination of bacterial endospores such as Bacillus anthracis has traditionally required the use of harsh or caustic chemicals. The aim of this study was to evaluate the efficacy of a chlorine dioxide decontaminant in killing Bacillus anthracis spores in solution and on a human skin simulant (porcine cadaver skin, compared to that of commonly used sodium hypochlorite or soapy water decontamination procedures. In addition, the relative toxicities of these decontaminants were compared in human skin keratinocyte primary cultures. The chlorine dioxide decontaminant was similarly effective to sodium hypochlorite in reducing spore numbers of Bacillus anthracis Ames in liquid suspension after a 10 minute exposure. After five minutes, the chlorine dioxide product was significantly more efficacious. Decontamination of isolated swine skin contaminated with Bacillus anthracis Sterne with the chlorine dioxide product resulted in no viable spores sampled. The toxicity of the chlorine dioxide decontaminant was up to two orders of magnitude less than that of sodium hypochlorite in human skin keratinocyte cultures. In summary, the chlorine dioxide based decontaminant efficiently killed Bacillus anthracis spores in liquid suspension, as well as on isolated swine skin, and was less toxic than sodium hypochlorite in cultures of human skin keratinocytes.

  12. The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

    Science.gov (United States)

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-06-02

    Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Bacillus anthracis infections – new possibilities of treatment

    Directory of Open Access Journals (Sweden)

    Dorota Żakowska

    2015-05-01

    Full Text Available [b]Introduction and objective[/b]. [i]Bacillus anthracis[/i] is one of biological agents which may be used in bioterrorism attacks. The aim of this study a review of the new treatment possibilities of anthrax, with particular emphasis on the treatment of pulmonary anthrax. [b]Abbreviated description of the state of knowledge[/b]. Pulmonary anthrax, as the most dangerous clinical form of the disease, is also extremely difficult to treat. Recently, considerable progress in finding new drugs and suitable therapy for anthrax has been achieved, for example, new antibiotics worth to mentioning, levofloxacin, daptomycin, gatifloxacin and dalbavancin. However, alternative therapeutic options should also be considered, among them the antimicrobial peptides, characterized by lack of inducible mechanisms of pathogen resistance. Very promising research considers bacteriophages lytic enzymes against selected bacteria species, including antibiotic-resistant strains. [b]Results[/b]. Interesting results were obtained using monoclonal antibodies: raxibacumab, cAb29 or cocktails of antibodies. The application of CpG oligodeoxynucleotides to boost the immune response elicited by Anthrax Vaccine Adsorbed and CMG2 protein complexes, also produced satisfying therapy results. Furthermore, the IFN-α and IFN-β, PA-dominant negative mutant, human inter-alpha inhibitor proteins and LF inhibitors in combination with ciprofloxacin, also showed very promising results. [b]Conclusions[/b]. Recently, progress has been achieved in inhalation anthrax treatment. The most promising new possibilities include: new antibiotics, peptides and bacteriophages enzymes, monoclonal antibodies, antigen PA mutants, and inter alpha inhibitors applications. In the case of the possibility of bioterrorist attacks, the examination of inhalation anthrax treatment should be intensively continued.

  14. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    Directory of Open Access Journals (Sweden)

    Ma’ayan Israeli

    2016-08-01

    Full Text Available Edema Factor (EF, the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP, and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules.

  15. clpC operon regulates cell architecture and sporulation in Bacillus anthracis.

    Science.gov (United States)

    Singh, Lalit K; Dhasmana, Neha; Sajid, Andaleeb; Kumar, Prasun; Bhaduri, Asani; Bharadwaj, Mitasha; Gandotra, Sheetal; Kalia, Vipin C; Das, Taposh K; Goel, Ajay K; Pomerantsev, Andrei P; Misra, Richa; Gerth, Ulf; Leppla, Stephen H; Singh, Yogendra

    2015-03-01

    The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knockout strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and open up further interest on this operon, which might be of importance to success of B. anthracis as pathogen. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  16. Real-Time PCR Assay for a Unique Chromosomal Sequence of Bacillus anthracis

    Science.gov (United States)

    2004-12-01

    13061 Neisseria lactamica .............................................................. 23970 Bacillus coagulans ...NEG Bacillus coagulane 7050 NEG NEG Bacillus cereus 13472 NEG NEG Bacillus licheniforms 12759 NEG NEG Bacillus cereus 13824 NEG NEG Bacillus ...Assay for a Unique Chromosomal Sequence of Bacillus anthracis Elizabeth Bode,1 William Hurtle,2† and David Norwood1* United States Army Medical

  17. Inactivation of Bacillus anthracis Spores during Laboratory-Scale Composting of Feedlot Cattle Manure

    Science.gov (United States)

    Xu, Shanwei; Harvey, Amanda; Barbieri, Ruth; Reuter, Tim; Stanford, Kim; Amoako, Kingsley K.; Selinger, Leonard B.; McAllister, Tim A.

    2016-01-01

    Anthrax outbreaks in livestock have social, economic and health implications, altering farmer’s livelihoods, impacting trade and posing a zoonotic risk. Our study investigated the survival of Bacillus thuringiensis and B. anthracis spores sporulated at 15, 20, or 37°C, over 33 days of composting. Spores (∼7.5 log10 CFU g-1) were mixed with manure and composted in laboratory scale composters. After 15 days, the compost was mixed and returned to the composter for a second cycle. Temperatures peaked at 71°C on day 2 and remained ≥55°C for an average of 7 days in the first cycle, but did not exceed 55°C in the second. For B. thuringiensis, spores generated at 15 and 21°C exhibited reduced (P composting for spores generated at 15, 21, and 37°C, respectively. For both species, spore viability declined more rapidly (P composting cycle. Our findings suggest that the duration of thermophilic exposure (≥55°C) is the main factor influencing survival of B. anthracis spores in compost. As sporulation temperature did not influence survival of B. anthracis, composting may lower the viability of spores associated with carcasses infected with B. anthracis over a range of sporulation temperatures. PMID:27303388

  18. The effect of growth medium on B. anthracis Sterne spore carbohydrate content.

    Science.gov (United States)

    Colburn, Heather A; Wunschel, David S; Antolick, Kathryn C; Melville, Angela M; Valentine, Nancy B

    2011-06-01

    The expressed characteristics of biothreat agents may be impacted by variations in the culture environment, including growth medium formulation. The carbohydrate composition of B. anthracis spores has been well studied, particularly for the exosporium, which is the outermost spore structure. The carbohydrate composition of the exosporium has been demonstrated to be distinct from the vegetative form containing unique monosaccharides. We have investigated the carbohydrate composition of B. anthracis Sterne spores produced using four different medium types formulated with different sources of medium components. The amount of rhamnose, 3-O-methyl rhamnose and galactosamine was found to vary significantly between spores cultured using different medium formulations. The relative abundance of these monosaccharides compared to other monosaccharides such as mannosamine was also found to vary with medium type. Specific medium components were also found to impact the carbohydrate profile. Xylose has not been previously described in B. anthracis spores but was detected at low levels in two media. This may represent residual material from the brewery yeast extract used to formulate these two media. These results illustrate the utility of this method to capture the impact of growth medium on carbohydrate variation in spores. Detecting carbohydrate profiles in B. anthracis evidentiary material may provide useful forensic information on the growth medium used for sporulation. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Atomic Structure and Nonhomologous End-Joining Function of the Polymerase Component of Bacterial DNA Ligase D

    Energy Technology Data Exchange (ETDEWEB)

    Zhu,H.; Nandakumar, J.; Aniukwu, J.; Wang, L.; Glickman, M.; Lima, C.; Shuman, S.

    2006-01-01

    DNA ligase D (LigD) is a large polyfunctional protein that participates in a recently discovered pathway of nonhomologous end-joining in bacteria. LigD consists of an ATP-dependent ligase domain fused to a polymerase domain (Pol) and a phosphoesterase module. The Pol activity is remarkable for its dependence on manganese, its ability to perform templated and nontemplated primer extension reactions, and its preference for adding ribonucleotides to blunt DNA ends. Here we report the 1.5- Angstroms crystal structure of the Pol domain of Pseudomonas LigD and its complexes with manganese and ATP-dATP substrates, which reveal a minimized polymerase with a two-metal mechanism and a fold similar to that of archaeal DNA primase. Mutational analysis highlights the functionally relevant atomic contacts in the active site. Although distinct nucleoside conformations and contacts for ATP versus dATP are observed in the cocrystals, the functional analysis suggests that the ATP-binding mode is the productive conformation for dNMP and rNMP incorporation. We find that a mutation of Mycobacterium LigD that uniquely ablates the polymerase activity results in increased fidelity of blunt-end double-strand break repair in vivo by virtue of eliminating nucleotide insertions at the recombination junctions. Thus, LigD Pol is a direct catalyst of mutagenic nonhomologous end-joining in vivo. Our studies underscore a previously uncharacterized role for the primase-like polymerase family in DNA repair.

  20. Impact of spores on the comparative efficacies of five antibiotics for treatment of Bacillus anthracis in an in vitro hollow fiber pharmacodynamic model.

    Science.gov (United States)

    Louie, Arnold; VanScoy, Brian D; Brown, David L; Kulawy, Robert W; Heine, Henry S; Drusano, George L

    2012-03-01

    Bacillus anthracis, the bacterium that causes anthrax, is an agent of bioterrorism. The most effective antimicrobial therapy for B. anthracis infections is unknown. An in vitro pharmacodynamic model of B. anthracis was used to compare the efficacies of simulated clinically prescribed regimens of moxifloxacin, linezolid, and meropenem with the "gold standards," doxycycline and ciprofloxacin. Treatment outcomes for isogenic spore-forming and non-spore-forming strains of B. anthracis were compared. Against spore-forming B. anthracis, ciprofloxacin, moxifloxacin, linezolid, and meropenem reduced the B. anthracis population by 4 log(10) CFU/ml over 10 days. Doxycycline reduced the population of this B. anthracis strain by 5 log(10) CFU/ml (analysis of variance [ANOVA] P = 0.01 versus other drugs). Against an isogenic non-spore-forming strain, meropenem killed the vegetative B. anthracis the fastest, followed by moxifloxacin and ciprofloxacin and then doxycycline. Linezolid offered the lowest bacterial kill rate. Heat shock studies using the spore-producing B. anthracis strain showed that with moxifloxacin, ciprofloxacin, and meropenem therapies the total population was mostly spores, while the population was primarily vegetative bacteria with linezolid and doxycycline therapies. Spores have a profound impact on the rate and extent of killing of B. anthracis. Against spore-forming B. anthracis, the five antibiotics killed the total (spore and vegetative) bacterial population at similar rates (within 1 log(10) CFU/ml of each other). However, bactericidal antibiotics killed vegetative B. anthracis faster than bacteriostatic drugs. Since only vegetative-phase B. anthracis produces the toxins that may kill the infected host, the rate and mechanism of killing of an antibiotic may determine its overall in vivo efficacy. Further studies are needed to examine this important observation.

  1. The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid.

    Science.gov (United States)

    Celebi, Ozgur; Buyuk, Fatih; Pottage, Tom; Crook, Ant; Hawkey, Suzanna; Cooper, Callum; Bennett, Allan; Sahin, Mitat; Baillie, Leslie

    2016-01-01

    Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 10(4) spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites.

  2. The use of germinants to potentiate the sensitivity of Bacillus anthracis spores to peracetic acid

    Directory of Open Access Journals (Sweden)

    Ozgur eCelebi

    2016-01-01

    Full Text Available Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM and inosine (5 mM to reduce the concentration of peracetic acid (PAA required to inactivate B.anthracis spores. While L-alanine significantly enhanced (p=0.0085 the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p=0.0009. To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B.anthracis to increase the level of contamination to 104 spores/g. Treatment with germinants followed one hour later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B.anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p<0.0001 in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B.anthracis spores contaminated sites.

  3. The search and identification of the new immunodiagnostic targets of bacillus anthracis spore

    International Nuclear Information System (INIS)

    Biketov, S.; Dunaytsev, I.; Baranova, E.; Marinin, L.; Dyatlov, I.

    2009-01-01

    Spores of Bacillus anthracis have been used as bio warfare agent to bio terrorize purposes. As efficiency of anti-epidemic measures included urgent prevention and treatment is determined by terms within which the bio agent is identified. Direct and rapid spore detection by antibodies based detection system is very attractive alternative to current PCR-based assays or routine phenotyping which are the most accurate but are also complex, time-consumption and expensive. The main difficulty with respect to such kind of anthrax spores detection is a cross-reaction with spores of closely related bacteria. For development of species-specific antibodies to anthrax spores recombinant scFvs or hybridoma technique were used. In both case surface spore antigens contained species-specific epitopes are need. Among exosporium proteins only ExsF(BxpB), ExsK and SoaA are specific to B.cereus group. On the surface of B. anthracis spores, a unique tetrasaccharides containing an novel monosaccharide - anthrose, was discovered. It was shown that anthrose can be serving as species-specific target for B. anthracis spores detection. We have revealed that EA1 isolated from spore of Russians strain STI-1 contain carbohydrate which formed species-specific epitopes and determine immunogenicity of this antigen. Antibodies to this antigen specifically recognized the surface target of B. anthracis spores and do not reacted with others Bacillus spore. Based on these antibodies we developed the test-systems in different formats for rapid direct detection and identification of B. anthracis spores. The results of trial these test-systems with using more than 50 different Bacillus strains were indicated that carbohydrate of EA1 isolated from spore is effective immunodiagnostic target for anthrax spores bio detection.(author)

  4. The Pathogenomic Sequence Analysis of B. cereus and B.thuringiensis Isolates Closely Related to Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Han, Cliff S.; Xie, Gary; Challacombe, Jean F.; Altherr, MichaelR.; Smriti, B.; Bruce, David; Campbell, Connie S.; Campbell, Mary L.; Chen, Jin; Chertkov, Olga; Cleland, Cathy; Dimitrijevic-Bussod, M.; Doggett, Norman A.; Fawcett, John J.; Glavina, Tijana; Goodwin, Lynne A.; Hill, Karen K.; Hitchcock, Penny; Jackson, Paul J.; Keim, Paul; Kewalramani, Avinash Ramesh; Longmire, Jon; Lucas, Susan; Malfatti,Stephanie; McMurry, Kim; Meincke, Linda J.; Misra, Monica; Moseman,Bernice L.; Mundt, Mark; Munk, A. Christine; Okinaka, Richard T.; Parson-Quintana, B.; Reilly, Lee P.; Richardson, Paul; Robinson, DonnaL.; Rubin, Eddy; Saunders, Elizabeth; Tapia, Roxanne; Tesmer, Judith G.; Thayer, Nina; Thompson, Linda S.; Tice, Hope; Ticknor, Lawrence O.; Wills, Patti L.; Gilna, Payl; Brettin, Thomas S.

    2005-08-18

    The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B.cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including Banthracis. Comparative analysis of these two genomes with other members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.

  5. Laboratory Studies on Surface Sampling of Bacillus anthracis Contamination: Summary, Gaps, and Recommendations

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F.; Amidan, Brett G.; Hu, Rebecca

    2011-11-28

    This report summarizes previous laboratory studies to characterize the performance of methods for collecting, storing/transporting, processing, and analyzing samples from surfaces contaminated by Bacillus anthracis or related surrogates. The focus is on plate culture and count estimates of surface contamination for swab, wipe, and vacuum samples of porous and nonporous surfaces. Summaries of the previous studies and their results were assessed to identify gaps in information needed as inputs to calculate key parameters critical to risk management in biothreat incidents. One key parameter is the number of samples needed to make characterization or clearance decisions with specified statistical confidence. Other key parameters include the ability to calculate, following contamination incidents, the (1) estimates of Bacillus anthracis contamination, as well as the bias and uncertainties in the estimates, and (2) confidence in characterization and clearance decisions for contaminated or decontaminated buildings. Gaps in knowledge and understanding identified during the summary of the studies are discussed and recommendations are given for future studies.

  6. Automated thermochemolysis reactor for detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Li, Dan [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States); Rands, Anthony D.; Losee, Scott C. [Torion Technologies, American Fork, UT 84003 (United States); Holt, Brian C. [Department of Statistics, Brigham Young University, Provo, UT 84602 (United States); Williams, John R. [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States); Lammert, Stephen A. [Torion Technologies, American Fork, UT 84003 (United States); Robison, Richard A. [Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602 (United States); Tolley, H. Dennis [Department of Statistics, Brigham Young University, Provo, UT 84602 (United States); Lee, Milton L., E-mail: milton_lee@byu.edu [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States)

    2013-05-02

    Graphical abstract: -- Highlights: •An automated sample preparation system for Bacillus anthracis endospores was developed. •A thermochemolysis method was applied to produce and derivatize biomarkers for Bacillus anthracis detection. •The autoreactor controlled the precise delivery of reagents, and TCM reaction times and temperatures. •Solid phase microextraction was used to extract biomarkers, and GC–MS was used for final identification. •This autoreactor was successfully applied to the identification of Bacillus anthracis endospores. -- Abstract: An automated sample preparation system was developed and tested for the rapid detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry (GC–MS) for eventual use in the field. This reactor is capable of automatically processing suspected bio-threat agents to release and derivatize unique chemical biomarkers by thermochemolysis (TCM). The system automatically controls the movement of sample vials from one position to another, crimping of septum caps onto the vials, precise delivery of reagents, and TCM reaction times and temperatures. The specific operations of introduction of sample vials, solid phase microextraction (SPME) sampling, injection into the GC–MS system, and ejection of used vials from the system were performed manually in this study, although they can be integrated into the automated system. Manual SPME sampling is performed by following visual and audible signal prompts for inserting the fiber into and retracting it from the sampling port. A rotating carousel design allows for simultaneous sample collection, reaction, biomarker extraction and analysis of sequential samples. Dipicolinic acid methyl ester (DPAME), 3-methyl-2-butenoic acid methyl ester (a fragment of anthrose) and two methylated sugars were used to compare the performance of the autoreactor with manual TCM. Statistical algorithms were used to construct reliable bacterial endospore signatures, and 24

  7. Automated thermochemolysis reactor for detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry

    International Nuclear Information System (INIS)

    Li, Dan; Rands, Anthony D.; Losee, Scott C.; Holt, Brian C.; Williams, John R.; Lammert, Stephen A.; Robison, Richard A.; Tolley, H. Dennis; Lee, Milton L.

    2013-01-01

    Graphical abstract: -- Highlights: •An automated sample preparation system for Bacillus anthracis endospores was developed. •A thermochemolysis method was applied to produce and derivatize biomarkers for Bacillus anthracis detection. •The autoreactor controlled the precise delivery of reagents, and TCM reaction times and temperatures. •Solid phase microextraction was used to extract biomarkers, and GC–MS was used for final identification. •This autoreactor was successfully applied to the identification of Bacillus anthracis endospores. -- Abstract: An automated sample preparation system was developed and tested for the rapid detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry (GC–MS) for eventual use in the field. This reactor is capable of automatically processing suspected bio-threat agents to release and derivatize unique chemical biomarkers by thermochemolysis (TCM). The system automatically controls the movement of sample vials from one position to another, crimping of septum caps onto the vials, precise delivery of reagents, and TCM reaction times and temperatures. The specific operations of introduction of sample vials, solid phase microextraction (SPME) sampling, injection into the GC–MS system, and ejection of used vials from the system were performed manually in this study, although they can be integrated into the automated system. Manual SPME sampling is performed by following visual and audible signal prompts for inserting the fiber into and retracting it from the sampling port. A rotating carousel design allows for simultaneous sample collection, reaction, biomarker extraction and analysis of sequential samples. Dipicolinic acid methyl ester (DPAME), 3-methyl-2-butenoic acid methyl ester (a fragment of anthrose) and two methylated sugars were used to compare the performance of the autoreactor with manual TCM. Statistical algorithms were used to construct reliable bacterial endospore signatures, and 24

  8. Impedance Measurements Could Accelerate Phage-Based Identification of Bacillus anthracis and Other Bacteria

    Science.gov (United States)

    2016-09-01

    Impedance Measurements Could Accelerate Phage-Based Identification of Bacillus anthracis And Other Bacteria Thomas Brown, Salwa Shan, Teresa...infection can be detected as early as one hour after exposing as few as 105 CFU bacteria to the stressor. We predicted that similar responses could be used... bacteria to form confluent growth and for phage-induced plaques to appear. Techniques that permit faster detection of species-specific bacteria /phage

  9. Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

    International Nuclear Information System (INIS)

    Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.; Liu, Shihui; Leppla, Stephen H.

    2013-01-01

    Highlights: ► Non-infectious and protease-deficient Bacillus anthracis protein expression system. ► Successful expression and purification of a tumor-targeted fusion protein drug. ► Very low endotoxin contamination of purified protein. ► Efficient protein secretion simplifies purification. ► Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

  10. Green-Tea and Epigallocatechin-3-Gallate are Bactericidal against Bacillus anthracis

    Science.gov (United States)

    2017-06-13

    strategies against B. anthracis (3). 60 After water, tea is the most consumed beverage in the world. Although containing little 61 caloric value, teas...Civilian B. 2002. Anthrax as a biological weapon, 2002: updated 270 recommendations for management . JAMA 287:2236-52. 271 4. Cabrera C, Artacho R...Sharma A, Gupta S, Sarethy IP, Dang S, Gabrani R. 2012. Green tea extract: possible mechanism 285 and antibacterial activity on skin pathogens. Food

  11. Evaluation of immunoradiometric and ELISA versions of a microtitre plate assay for Bacillus anthracis spores

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, A P; Martin, K L; Cross, N L [Chemical Defence Experimental Establishment, Porton (UK); Drake, R G [Glasgow Univ. (UK). Inst. of Biochemistry

    1984-05-11

    Solid-phase indirectly-labelled antibody assays for Bacillus anthracis spores heat-fixed on polystyrene microtitre plates were compared as immunoradiometric assay (IRMA) and enzyme-linked immunosorbent assay (ELISA) versions. Signal-to-noise ratios were usually higher in the IRMA than in the ELISA performed under parallel conditions but replicates were more varied in the IRMA. The antigen detection threshold and resolution limit calculated after regression analysis were broadly comparable in the 2 types of assay.

  12. Modeling the potential distribution of Bacillus anthracis under multiple climate change scenarios for Kazakhstan.

    Directory of Open Access Journals (Sweden)

    Timothy Andrew Joyner

    Full Text Available Anthrax, caused by the bacterium Bacillus anthracis, is a zoonotic disease that persists throughout much of the world in livestock, wildlife, and secondarily infects humans. This is true across much of Central Asia, and particularly the Steppe region, including Kazakhstan. This study employed the Genetic Algorithm for Rule-set Prediction (GARP to model the current and future geographic distribution of Bacillus anthracis in Kazakhstan based on the A2 and B2 IPCC SRES climate change scenarios using a 5-variable data set at 55 km(2 and 8 km(2 and a 6-variable BioClim data set at 8 km(2. Future models suggest large areas predicted under current conditions may be reduced by 2050 with the A2 model predicting approximately 14-16% loss across the three spatial resolutions. There was greater variability in the B2 models across scenarios predicting approximately 15% loss at 55 km(2, approximately 34% loss at 8 km(2, and approximately 30% loss with the BioClim variables. Only very small areas of habitat expansion into new areas were predicted by either A2 or B2 in any models. Greater areas of habitat loss are predicted in the southern regions of Kazakhstan by A2 and B2 models, while moderate habitat loss is also predicted in the northern regions by either B2 model at 8 km(2. Anthrax disease control relies mainly on livestock vaccination and proper carcass disposal, both of which require adequate surveillance. In many situations, including that of Kazakhstan, vaccine resources are limited, and understanding the geographic distribution of the organism, in tandem with current data on livestock population dynamics, can aid in properly allocating doses. While speculative, contemplating future changes in livestock distributions and B. anthracis spore promoting environments can be useful for establishing future surveillance priorities. This study may also have broader applications to global public health surveillance relating to other diseases in addition to B

  13. Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.; Liu, Shihui [National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States); Leppla, Stephen H., E-mail: sleppla@niaid.nih.gov [National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Non-infectious and protease-deficient Bacillus anthracis protein expression system. Black-Right-Pointing-Pointer Successful expression and purification of a tumor-targeted fusion protein drug. Black-Right-Pointing-Pointer Very low endotoxin contamination of purified protein. Black-Right-Pointing-Pointer Efficient protein secretion simplifies purification. Black-Right-Pointing-Pointer Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGF{alpha}). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGF{alpha}). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

  14. The human-bacterial pathogen protein interaction networks of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Matthew D Dyer

    2010-08-01

    Full Text Available Bacillus anthracis, Francisella tularensis, and Yersinia pestis are bacterial pathogens that can cause anthrax, lethal acute pneumonic disease, and bubonic plague, respectively, and are listed as NIAID Category A priority pathogens for possible use as biological weapons. However, the interactions between human proteins and proteins in these bacteria remain poorly characterized leading to an incomplete understanding of their pathogenesis and mechanisms of immune evasion.In this study, we used a high-throughput yeast two-hybrid assay to identify physical interactions between human proteins and proteins from each of these three pathogens. From more than 250,000 screens performed, we identified 3,073 human-B. anthracis, 1,383 human-F. tularensis, and 4,059 human-Y. pestis protein-protein interactions including interactions involving 304 B. anthracis, 52 F. tularensis, and 330 Y. pestis proteins that are uncharacterized. Computational analysis revealed that pathogen proteins preferentially interact with human proteins that are hubs and bottlenecks in the human PPI network. In addition, we computed modules of human-pathogen PPIs that are conserved amongst the three networks. Functionally, such conserved modules reveal commonalities between how the different pathogens interact with crucial host pathways involved in inflammation and immunity.These data constitute the first extensive protein interaction networks constructed for bacterial pathogens and their human hosts. This study provides novel insights into host-pathogen interactions.

  15. Optimization of a sample processing protocol for recovery of Bacillus anthracis spores from soil

    Science.gov (United States)

    Silvestri, Erin E.; Feldhake, David; Griffin, Dale; Lisle, John T.; Nichols, Tonya L.; Shah, Sanjiv; Pemberton, A; Schaefer III, Frank W

    2016-01-01

    Following a release of Bacillus anthracis spores into the environment, there is a potential for lasting environmental contamination in soils. There is a need for detection protocols for B. anthracis in environmental matrices. However, identification of B. anthracis within a soil is a difficult task. Processing soil samples helps to remove debris, chemical components, and biological impurities that can interfere with microbiological detection. This study aimed to optimize a previously used indirect processing protocol, which included a series of washing and centrifugation steps. Optimization of the protocol included: identifying an ideal extraction diluent, variation in the number of wash steps, variation in the initial centrifugation speed, sonication and shaking mechanisms. The optimized protocol was demonstrated at two laboratories in order to evaluate the recovery of spores from loamy and sandy soils. The new protocol demonstrated an improved limit of detection for loamy and sandy soils over the non-optimized protocol with an approximate matrix limit of detection at 14 spores/g of soil. There were no significant differences overall between the two laboratories for either soil type, suggesting that the processing protocol will be robust enough to use at multiple laboratories while achieving comparable recoveries.

  16. Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Morgunova, Ekaterina [Karolinska Institutet NOVUM, Center of Structural Biochemistry, Hälsovägen 7-9, 141 57 Huddinge (Sweden); Illarionov, Boris; Saller, Sabine [Institut für Lebensmittelchemie, Universität Hamburg, Grindelallee 117, 20146 Hamburg (Germany); Popov, Aleksander [European Synchrotron Radiation Facility, BP 220, F-38043 Grenoble CEDEX 09 (France); Sambaiah, Thota [Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University (United States); Bacher, Adelbert [Chemistry Department, Technical University of Munich, 85747 Garching (Germany); Cushman, Mark [Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University (United States); Fischer, Markus [Institut für Lebensmittelchemie, Universität Hamburg, Grindelallee 117, 20146 Hamburg (Germany); Ladenstein, Rudolf, E-mail: rudolf.ladenstein@ki.se [Karolinska Institutet NOVUM, Center of Structural Biochemistry, Hälsovägen 7-9, 141 57 Huddinge (Sweden)

    2010-09-01

    Crystallographic studies of lumazine synthase, the penultimate enzyme of the riboflavin-biosynthetic pathway in B. anthracis, provide a structural framework for the design of antibiotic inhibitors, together with calorimetric and kinetic investigations of inhibitor binding. The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to R{sub cryst} = 23.7% (R{sub free} = 28.4%) at a resolution of 3.5 Å. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis.

  17. Storage Effects on Sample Integrity of Environmental Surface Sampling Specimens with Bacillus anthracis Spores.

    Science.gov (United States)

    Perry, K Allison; O'Connell, Heather A; Rose, Laura J; Noble-Wang, Judith A; Arduino, Matthew J

    The effect of packaging, shipping temperatures and storage times on recovery of Bacillus anthracis . Sterne spores from swabs was investigated. Macrofoam swabs were pre-moistened, inoculated with Bacillus anthracis spores, and packaged in primary containment or secondary containment before storage at -15°C, 5°C, 21°C, or 35°C for 0-7 days. Swabs were processed according to validated Centers for Disease Control/Laboratory Response Network culture protocols, and the percent recovery relative to a reference sample (T 0 ) was determined for each variable. No differences were observed in recovery between swabs held at -15° and 5°C, (p ≥ 0.23). These two temperatures provided significantly better recovery than swabs held at 21°C or 35°C (all 7 days pooled, p ≤ 0.04). The percent recovery at 5°C was not significantly different if processed on days 1, 2 or 4, but was significantly lower on day 7 (day 2 vs. 7, 5°C, 10 2 , p=0.03). Secondary containment provided significantly better percent recovery than primary containment, regardless of storage time (5°C data, p ≤ 0.008). The integrity of environmental swab samples containing Bacillus anthracis spores shipped in secondary containment was maintained when stored at -15°C or 5°C and processed within 4 days to yield the optimum percent recovery of spores.

  18. Rap phosphatase of virulence plasmid pXO1 inhibits Bacillus anthracis sporulation.

    Science.gov (United States)

    Bongiorni, Cristina; Stoessel, Ricarda; Shoemaker, Dorinda; Perego, Marta

    2006-01-01

    This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis.

  19. Rap Phosphatase of Virulence Plasmid pXO1 Inhibits Bacillus anthracis Sporulation†

    Science.gov (United States)

    Bongiorni, Cristina; Stoessel, Ricarda; Shoemaker, Dorinda; Perego, Marta

    2006-01-01

    This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis. PMID:16385039

  20. Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy.

    Science.gov (United States)

    Carlsson, Emil; Thwaite, Joanne E; Jenner, Dominic C; Spear, Abigail M; Flick-Smith, Helen; Atkins, Helen S; Byrne, Bernadette; Ding, Jeak Ling

    2016-01-01

    Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence.

  1. A Dual Role for the Bacillus anthracis Master Virulence Regulator AtxA: Control of Sporulation and Anthrax Toxin Production.

    Science.gov (United States)

    Dale, Jennifer L; Raynor, Malik J; Ty, Maureen C; Hadjifrangiskou, Maria; Koehler, Theresa M

    2018-01-01

    Bacillus anthracis is an endemic soil bacterium that exhibits two different lifestyles. In the soil environment, B. anthracis undergoes a cycle of saprophytic growth, sporulation, and germination. In mammalian hosts, the pathogenic lifestyle of B. anthracis is spore germination followed by vegetative cell replication, but cells do not sporulate. During infection, and in specific culture conditions, transcription of the structural genes for the anthrax toxin proteins and the biosynthetic operon for capsule synthesis is positively controlled by the regulatory protein AtxA. A critical role for the atxA gene in B. anthracis virulence has been established. Here we report an inverse relationship between toxin production and sporulation that is linked to AtxA levels. During culture in conditions favoring sporulation, B. anthracis produces little to no AtxA. When B. anthracis is cultured in conditions favoring toxin gene expression, AtxA is expressed at relatively high levels and sporulation rate and efficiency are reduced. We found that a mutation within the atxA promoter region resulting in AtxA over-expression leads to a marked sporulation defect. The sporulation phenotype of the mutant is dependent upon pXO2-0075 , an atxA -regulated open reading frame located on virulence plasmid pXO2. The predicted amino acid sequence of the pXO2-0075 protein has similarity to the sensor domain of sporulation sensor histidine kinases. It was shown previously that pXO2-0075 overexpression suppresses sporulation. We have designated pXO2-0075 " skiA " for "sporulation kinase inhibitor." Our results indicate that in addition to serving as a positive regulator of virulence gene expression, AtxA modulates B. anthracis development.

  2. Enzymes involved in organellar DNA replication in photosynthetic eukaryotes.

    Science.gov (United States)

    Moriyama, Takashi; Sato, Naoki

    2014-01-01

    Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana, the replication-related enzymes of plastids and mitochondria are similar because many of them are dual targeted to both organelles, whereas in the red alga Cyanidioschyzon merolae, plastids and mitochondria contain different replication machinery components. The enzymes involved in organellar genome replication in green plants and red algae were derived from different origins, including proteobacterial, cyanobacterial, and eukaryotic lineages. In the present review, we summarize the available data for enzymes related to organellar genome replication in green plants and red algae. In addition, based on the type and distribution of replication enzymes in photosynthetic eukaryotes, we discuss the transitional history of replication enzymes in the organelles of plants.

  3. Amplification of the Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 lytic origin of DNA replication is dependent upon a cis-acting AT-rich region and an ORF50 response element and the trans-acting factors ORF50 (K-Rta) and K8 (K-bZIP)

    International Nuclear Information System (INIS)

    AuCoin, David P.; Colletti, Kelly S.; Cei, Sylvia A.; Papouskova, Iva; Tarrant, Margaret; Pari, Gregory S.

    2004-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), has significant sequence homology to Epstein-Barr virus (EBV). In cell culture, HHV8 is primarily latent, and viral genes associated with lytic replication are not expressed. Two lytic origins of DNA replication (oriLyt) are present within the HHV8 genome and are composed of an AT-rich region adjacent to GC-rich DNA sequences. We have now identified essential cis- and trans-acting elements required for oriLyt-dependent DNA replication. The transient replication assay was used to show that two AT-rich elements, three consensus AP1 transcription factor-binding sites, an ORF50 response element (RE), and a consensus TATA box motif are essential for efficient origin-dependent DNA replication. Transient transfection of luciferase reporter constructs indicated that the downstream region of the HHV8 oriLyt responds to ORF50 and suggests that part of the oriLyt may be an enhancer/promoter. In addition, a transient cotransfection-replication assay elucidated the set of trans-acting factors required for lytic DNA replication. These factors consist of homologues to the core replication proteins: ORF6 (ssDNA binding protein), ORF9 (DNA polymerase), ORF40-41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor) common to all herpesviruses along with ORF50 (K-Rta) and K8 (K-bZIP)

  4. Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: evidence for bypass of pyrimidine photodimers

    International Nuclear Information System (INIS)

    Livneh, Z.

    1986-01-01

    Replication of UV-irradiated circular single-stranded phage M13 DNA by Escherichia coli RNA polymerase (EC 2.7.7.6) and DNA polymerase III holoenzyme (EC 2.7.7.7) in the presence of single-stranded DNA binding protein yielded full-length as well as partially replicated products. A similar result was obtained with phage G4 DNA primed with E. coli DNA primase, and phage phi X174 DNA primed with a synthetic oligonucleotide. The fraction of full-length DNA was several orders of magnitude higher than predicted if pyrimidine photodimers were to constitute absolute blocks to DNA replication. Recent models have suggested that pyrimidine photodimers are absolute blocks to DNA replication and that SOS-induced proteins are required to allow their bypass. Our results demonstrate that, under in vitro replication conditions, E. coli DNA polymerase III holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins

  5. In vitro evaluation of the effect of linezolid and levofloxacin on Bacillus anthracis toxin production, spore formation and cell growth.

    Science.gov (United States)

    Head, Breanne M; Alfa, Michelle; Sitar, Daniel S; Rubinstein, Ethan; Meyers, Adrienne F A

    2017-02-01

    Owing to its ability to form spores and toxins, Bacillus anthracis is considered a bioterror agent. Although current therapeutic strategies can be effective, treatment does not prevent sporulation and toxin production. To quantify the combined effect of a protein synthesis inhibitor and a bactericidal agent on B. anthracis toxin production, sporulation and cell growth. Susceptibility and synergy titrations were conducted on B. anthracis Sterne and 03-0191 strains using linezolid and levofloxacin. The effect of antibiotic exposure on cell viability was evaluated using a continuous medium replacement model. In vitro static models were used to study the effect of linezolid and levofloxacin on sporulation and toxin production. Spores were quantified using the heat shock method. Toxin was quantified via commercial ELISA. Synergy titrations indicated that the combination was synergistic or indifferent; however, in all models antagonism was observed. In the spore model, linezolid resulted in the lowest sporulation rates, while combination therapy resulted in the highest. In the toxin model, linezolid prevented toxin production altogether. This study advances our understanding of the effects of combination therapy on B. anthracis infection. Used alone, linezolid therapy abolishes toxin production and reduces sporulation. These results suggest that studies using a step-wise approach using linezolid initially to stop sporulation and toxin production followed by levofloxacin to rapidly kill vegetative B. anthracis can be recommended. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. The reporting of a Bacillus anthracis B-clade strain in South Africa after more than 20 years.

    Science.gov (United States)

    Lekota, K E; Hassim, A; Rogers, P; Dekker, E H; Last, R; de Klerk-Lorist, L; van Heerden, H

    2018-05-02

    Anthrax is a disease with an age old history in Africa caused by the Gram-positive endospore forming soil bacterium Bacillus anthracis. Epizootics of wild ungulates occur annually in the enzootic region of Pafuri, Kruger National Park (KNP) in the Limpopo Province of South Africa. Rigorous routine surveillance and diagnostics in KNP, has not revealed these rare isolates since the 1990s, despite unabated annual outbreaks. In 2011 a cheetah was diagnosed as anthrax positive from a private game reserve in Limpopo Province and reported to State Veterinary Services for further investigation. Isolation, molecular diagnostics, whole genome sequencing and comparative genomics were carried out for B. anthracis KC2011. Bacteriological and molecular diagnostics confirmed the isolate as B. anthracis. Subsequent typing and whole genome single nucleotide polymorphisms analysis indicated it clustered alongside B. anthracis SA A0091 in the B.Br.010 SNP branch. Unlike B. anthracis KrugerB strain, KC2011 strain has unique SNPs and represents a new branch in the B-clade. The isolation and genotypic characterisation of KC2011 demonstrates a gap in the reporting of anthrax outbreaks in the greater Limpopo province area. The identification of vulnerable and susceptible cheetah mortalities due to this strain has implications for conservation measures and disease control.

  7. Bacillus cereus Biovar Anthracis Causing Anthrax in Sub-Saharan Africa-Chromosomal Monophyly and Broad Geographic Distribution.

    Directory of Open Access Journals (Sweden)

    Kym S Antonation

    2016-09-01

    Full Text Available Through full genome analyses of four atypical Bacillus cereus isolates, designated B. cereus biovar anthracis, we describe a distinct clade within the B. cereus group that presents with anthrax-like disease, carrying virulence plasmids similar to those of classic Bacillus anthracis. We have isolated members of this clade from different mammals (wild chimpanzees, gorillas, an elephant and goats in West and Central Africa (Côte d'Ivoire, Cameroon, Central African Republic and Democratic Republic of Congo. The isolates shared several phenotypic features of both B. anthracis and B. cereus, but differed amongst each other in motility and their resistance or sensitivity to penicillin. They all possessed the same mutation in the regulator gene plcR, different from the one found in B. anthracis, and in addition, carry genes which enable them to produce a second capsule composed of hyaluronic acid. Our findings show the existence of a discrete clade of the B. cereus group capable of causing anthrax-like disease, found in areas of high biodiversity, which are possibly also the origin of the worldwide distributed B. anthracis. Establishing the impact of these pathogenic bacteria on threatened wildlife species will require systematic investigation. Furthermore, the consumption of wildlife found dead by the local population and presence in a domestic animal reveal potential sources of exposure to humans.

  8. Mitochondrial DNA replication: a PrimPol perspective

    Science.gov (United States)

    Bailey, Laura J.

    2017-01-01

    PrimPol, (primase–polymerase), the most recently identified eukaryotic polymerase, has roles in both nuclear and mitochondrial DNA maintenance. PrimPol is capable of acting as a DNA polymerase, with the ability to extend primers and also bypass a variety of oxidative and photolesions. In addition, PrimPol also functions as a primase, catalysing the preferential formation of DNA primers in a zinc finger-dependent manner. Although PrimPol's catalytic activities have been uncovered in vitro, we still know little about how and why it is targeted to the mitochondrion and what its key roles are in the maintenance of this multicopy DNA molecule. Unlike nuclear DNA, the mammalian mitochondrial genome is circular and the organelle has many unique proteins essential for its maintenance, presenting a differing environment within which PrimPol must function. Here, we discuss what is currently known about the mechanisms of DNA replication in the mitochondrion, the proteins that carry out these processes and how PrimPol is likely to be involved in assisting this vital cellular process. PMID:28408491

  9. Enhanced production and purification of recombinant surface array protein (Sap) for use in detection of Bacillus anthracis.

    Science.gov (United States)

    Puranik, Nidhi; Tripathi, N K; Pal, V; Goel, Ajay Kumar

    2018-05-01

    Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis , which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli . The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively. The protein purity and its reactivity were confirmed employing SDS-PAGE and Western blot, respectively. The antibodies raised against purified Sap were evaluated by Western blotting for detection of Sap released by B. anthracis . Our results showed that the Sap could be a novel marker for detection and confirmation of B. anthracis .

  10. Effects of benzo[a]pyrene-DNA adducts on a reconstituted replication system

    International Nuclear Information System (INIS)

    Brown, W.C.; Romano, L.J.

    1991-01-01

    The authors have used a partially reconstituted replication system consisting of T7 DNA polymerase and T7 gene 4 protein to examine the effect of benzo[a]pyrene (B[a]P) adducts on DNA synthesis and gene 4 protein activities. The gene 4 protein is required for T7 DNA replication because of its ability to act as both a primase and helicase. They show here that total synthesis decreases as the level of adducts per molecule of DNA increases, suggesting that the B[a]P adducts are blocking an aspect of the replication process. By challenging synthesis on oligonucleotide-primed B[a]P-modified DNA with unmodified DNA, they present evidence that the T7 DNA polymerase freely dissociates after encountering an adduct. Prior studies have shown that the gene 4 protein alone does not dissociate from the template during translocation upon encountering an adduct. However, when gene 4 protein primed DNA synthesis is challenged, they observe an increase in synthesis but to a lesser extent than observed on oligonucleotide-primed synthesis. Finally, they have examined DNA synthesis on duplex templates and show the B[a]P adducts inhibit synthesis by the T7 DNA polymerase and gene 4 protein to the same extent regardless of whether the adducts are positioned in the leading or lagging strand, while synthesis by the polymerase alone is inhibited only when the adducts are in the template strand

  11. Bacillus anthracis: una mirada molecular a un patógeno célebre Bacillus anthracis: a molecular look at a famous pathogen

    Directory of Open Access Journals (Sweden)

    María E Pavan

    2011-12-01

    Full Text Available Bacillus anthracis es un bacilo gram positivo del grupo Bacillus cereus, que posee un genoma extremadamente monomórfco y comparte gran similitud fsiológica y de estructura genética con B. cereus y Bacillus thuringiensis. En este artículo se describen nuevos métodos moleculares para la identifcación y tipifcación de B. anthracis, basados en repeticiones en tándem de número variable o en diferencias genéticas detectadas por secuenciación, desarrollados en los últimos años. Los aspectos moleculares de los factores de virulencia tradicionales, cápsula, antígeno protector, factor letal y factor edema se describen en profundidad, junto con factores de virulencia recientemente propuestos, como los sideróforos, petrobactina y bacilibactina, la adhesina de la capa S y la lipoproteína MntA. También se detalla la organización molecular de los megaplásmidos pXO1 y pXO2, incluyendo la isla de patogenicidad de pXO1. El esqueleto genético de estos plásmidos se ha encontrado en otras especies relacionadas, probablemente debido a eventos de transferencia lateral. Finalmente, se presentan los dos receptores celulares del antígeno protector, ANTXR1/TEM8 y ANTXR2/CMG2, esenciales en la interacción del patógeno con el hospedador. Los estudios moleculares realizados en los últimos años han permitido aumentar enormemente el conocimiento de los diferentes aspectos de este microorganismo y su relación con el hospedador, pero a la vez han abierto nuevos interrogantes sobre este notorio patógeno.Bacillus anthracis, a gram-positive rod belonging to the Bacillus cereus group, has an extremely monomorphic genome, and presents high structural and physiological similarity with B. cereus and Bacillus thuringiensis. In this work, the new molecular methods for the identifcation and typing of B. anthracis developed in the last years, based on variable number tandem repeats or on genetic differences detected through sequencing, are described. The

  12. Protein profiles of field isolates ofBacillus anthracis from different endemic areas of Indonesia

    Directory of Open Access Journals (Sweden)

    M Bhakti Poerwadikarta

    1998-03-01

    Full Text Available Sonicated cell-free extract proteins of 14 field isolates ofBacillus anthracis from six different endemic areas of Indonesia were analyzed by the use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE methods . The protein profiles of each field isolate tested demonstrated slightly different at the protein bands with molecular weights of 18, 37, 52, 65 and 70 kDa, and varied between the field isolates and vaccine strains. The variation could provide clues to the source of anthrax transmission whether it was originated from similar strain or not.

  13. Modeling the Ecological Niche of Bacillus anthracis to Map Anthrax Risk in Kyrgyzstan.

    Science.gov (United States)

    Blackburn, Jason K; Matakarimov, Saitbek; Kozhokeeva, Sabira; Tagaeva, Zhyldyz; Bell, Lindsay K; Kracalik, Ian T; Zhunushov, Asankadyr

    2017-03-01

    AbstractAnthrax, caused by the environmental bacterium Bacillus anthracis , is an important zoonosis nearly worldwide. In Central Asia, anthrax represents a major veterinary and public health concern. In the Republic of Kyrgyzstan, ongoing anthrax outbreaks have been reported in humans associated with handling infected livestock and contaminated animal by-products such as meat or hides. The current anthrax situation has prompted calls for improved insights into the epidemiology, ecology, and spatial distribution of the disease in Kyrgyzstan to better inform control and surveillance. Disease control for both humans and livestock relies on annual livestock vaccination ahead of outbreaks. Toward this, we used a historic database of livestock anthrax reported from 1932 to 2006 mapped at high resolution to develop an ecological niche model-based prediction of B. anthracis across Kyrgyzstan and identified spatial clusters of livestock anthrax using a cluster morphology statistic. We also defined the seasonality of outbreaks in livestock. Cattle were the most frequently reported across the time period, with the greatest number of cases in late summer months. Our niche models defined four areas as suitable to support pathogen persistence, the plateaus near Talas and Bishkek, the valleys of western Kyrgyzstan along the Fergana Valley, and the low-lying areas along the shore of Lake Isyk-Kul. These areas should be considered "at risk" for livestock anthrax and subsequent human cases. Areas defined by the niche models can be used to prioritize anthrax surveillance and inform efforts to target livestock vaccination campaigns.

  14. Rapid Detection of Bacillus anthracis Spores Using Immunomagnetic Separation and Amperometry

    Directory of Open Access Journals (Sweden)

    David F. Waller

    2016-12-01

    Full Text Available Portable detection and quantitation methods for Bacillus anthracis (anthrax spores in pure culture or in environmental samples are lacking. Here, an amperometric immunoassay has been developed utilizing immunomagnetic separation to capture the spores and remove potential interferents from test samples followed by amperometric measurement on a field-portable instrument. Antibody-conjugated magnetic beads and antibody-conjugated glucose oxidase were used in a sandwich format for the capture and detection of target spores. Glucose oxidase activity of spore pellets was measured indirectly via amperometry by applying a bias voltage after incubation with glucose, horseradish peroxidase, and the electron mediator 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid. Target capture was mediated by polyclonal antisera, whereas monoclonal antibodies were used for signal generation. This strategy maximized sensitivity (500 target spores, 5000 cfu/mL, while also providing a good specificity for Bacillus anthracis spores. Minimal signal deviation occurs in the presence of environmental interferents including soil and modified pH conditions, demonstrating the strengths of immunomagnetic separation. The simultaneous incubation of capture and detection antibodies and rapid substrate development (5 min result in short sample-to-signal times (less than an hour. With attributes comparable or exceeding that of ELISA and LFDs, amperometry is a low-cost, low-weight, and practical method for detecting anthrax spores in the field.

  15. [An historical, sociocultural view and in the fiction literature of Bacillus anthracis cases by shaving brushes].

    Science.gov (United States)

    Vázquez-Espinosa, E; Laganà, C; Vazquez, F

    2018-06-01

    In the period from 1915 to 1924 anthrax outbreaks were described by Bacillus anthracis due to the contamination of razor brushes that reached Europe and the United States from areas such as Japan, China or Russia. The brushes were made with badger hair, and then, to reduce the cost with horse hair and other animals. World War I supoosed that the traffics of these brushes, that passed through Europe, changed and the processes of sterilization of the same were deficient giving rise to these outbreaks, that in a percentage of 20% produced the death of the users. The impact of the fashion of wearing a beard, the presence of these cases in the press, in the society of that period, and literature are studied through the work of Agatha Christie who wrote, in 1936, the Hercules Poirot´s novel Cards on the table, and where she describes the murder of one of the characters with the shaving brush contaminated with Bacillus anthracis spores. ©The Author 2018. Published by Sociedad Española de Quimioterapia. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)(https://creativecommons.org/licenses/by-nc/4.0/).

  16. Identification of the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available Immunoproteomics was used to screen the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R. The spore and vegetative proteins were separated by 2D gel electrophoresis and transferred to polyvinylidene difluoride membranes, and then western blotting was performed with rabbit immune serum against B.anthracis live spores. Immunogenic spots were cut and digested by trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed to identify the proteins. As a result, 11 and 45 immunogenic proteins were identified in the spores and vegetative cells, respectively; 26 of which have not been reported previously. To verify their immunogenicity, 12 of the identified proteins were selected to be expressed, and the immune sera from the mice vaccinated by the 12 expressed proteins, except BA0887, had a specific western blot band with the A16R whole cellular lytic proteins. Some of these immunogenic proteins might be used as novel vaccine candidates themselves or for enhancing the protective efficacy of a protective-antigen-based vaccine.

  17. A Study on molecular characterization of Razi Bacillus anthracis Sterne 34F2 substrain in Iran

    Directory of Open Access Journals (Sweden)

    Tadayon, K.

    2016-07-01

    Full Text Available Anthrax, a zoonotic disease caused by Bacillus anthracis, has affected humans since ancient times. For genomic characterization of Razi B. anthracis Sterne 34F2 substrain, single nucleotide polymorphism (SNP genotyping method developed by Van Erth, variable-number tandem-repeat (VNTR-8 analysis proposed by Keim, and multiple-locus VNTR analysis (MLVA-3 introduced by Levy were employed. In the SNPs typing system, where the nucleotide content of the genome at 13 evolutionary canonical loci was collectively analyzed, the originally South African 34F2 substrain was categorized in the A.Br.001/002 subgroup. In the VNTR-8 analysis, fragments with lengths of 314, 229, 162, 580, 532, 158, and 137 bp were identified at the following loci: vrrA, vrrB1, vrrB2, vrrC1, vrrC2, CG3, and pxO1, respectively. In addition, application of Levy's MLVA-3 genotyping method revealed that the genome of this strain carried 941, 451, and 864 bp fragments at AA03, AJ03, and AA07 loci, respectively. The present findings are undoubtedly helpful in meeting the requirements set by the World Organization for Animal Health (OIE and World Health Organization (WHO for anthrax vaccine manufacturers including Razi Institute. However, further similar studies are required to promote the current epidemiological knowledge of anthrax in Iran.

  18. Sporulation and germination gene expression analysis of Bacillus anthracis Sterne spores in skim milk under heat and different intervention techniques

    Science.gov (United States)

    To investigate how B. anthracis Stene spores survive in milk under heat (80 degree C, 10 minutes), pasteurization (72 degree C, 15 seconds) and pasteurization plus microfiltration, the expression levels of genes that related to sporulation and germination were tested using real-time PCR assays. Tw...

  19. Pilot-scale crossflow-microfiltration and pasturization to remove spores of Bacillus anthracis (Sterne) from milk

    Science.gov (United States)

    HTST pasteurization of milk is generally ineffective against spore-forming bacteria such as Bacillus anthracis (BA) but is lethal to its vegetative cells. Crossflow microfiltration (MF), using ceramic membranes with a pore diameter of 1.4 um, has been shown to physically remove somatic cells, vegeta...

  20. Reagent-free and portable detection of Bacillus anthracis spores using a microfluidic incubator and smartphone microscope.

    Science.gov (United States)

    Hutchison, Janine R; Erikson, Rebecca L; Sheen, Allison M; Ozanich, Richard M; Kelly, Ryan T

    2015-09-21

    Bacillus anthracis is the causative agent of anthrax and can be contracted by humans and herbivorous mammals by inhalation, ingestion, or cutaneous exposure to bacterial spores. Due to its stability and disease potential, B. anthracis is a recognized biothreat agent and robust detection and viability methods are needed to identify spores from unknown samples. Here we report the use of smartphone-based microscopy (SPM) in combination with a simple microfluidic incubation device (MID) to detect 50 to 5000 B. anthracis Sterne spores in 3 to 5 hours. This technique relies on optical monitoring of the conversion of the ∼1 μm spores to the filamentous vegetative cells that range from tens to hundreds of micrometers in length. This distinguishing filament formation is unique to B. anthracis as compared to other members of the Bacillus cereus group. A unique feature of this approach is that the sample integrity is maintained, and the vegetative biomass can be removed from the chip for secondary molecular analysis such as PCR. Compared with existing chip-based and rapid viability PCR methods, this new approach reduces assay time by almost half, and is highly sensitive, specific, and cost effective.

  1. Crystal structure of B acillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity: AtxA multimerization, phosphorylation and activity

    Energy Technology Data Exchange (ETDEWEB)

    Hammerstrom, Troy G.; Lori, Horton B.; Swick, Michelle C.; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M.

    2014-12-30

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (HisAsp) and phosphoablative (HisAla) amino acid changes for activity in B.anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.

  2. Essential and non-essential DNA replication genes in the model halophilic Archaeon, Halobacterium sp. NRC-1

    Directory of Open Access Journals (Sweden)

    DasSarma Shiladitya

    2007-06-01

    Full Text Available Abstract Background Information transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes. Functional assignments of archaeal DNA replication genes have been primarily based upon sequence homology and biochemical studies of replisome components, but few genetic studies have been conducted thus far. We have developed a tractable genetic system for knockout analysis of genes in the model halophilic archaeon, Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential. Results Using a directed in-frame gene knockout method in Halobacterium sp. NRC-1, we examined nineteen genes predicted to be involved in DNA replication. Preliminary bioinformatic analysis of the large haloarchaeal Orc/Cdc6 family, related to eukaryotic Orc1 and Cdc6, showed five distinct clades of Orc/Cdc6 proteins conserved in all sequenced haloarchaea. Of ten orc/cdc6 genes in Halobacterium sp. NRC-1, only two were found to be essential, orc10, on the large chromosome, and orc2, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, polB1, and the chromosomally encoded euryarchaeal-specific D family, polD1/D2 (formerly called polA1/polA2 in the Halobacterium sp. NRC-1 genome sequence. The pNRC200-encoded B family polymerase, polB2, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, mcm, the eukaryotic-type DNA primase, pri1/pri2, the DNA polymerase sliding clamp, pcn, and the flap endonuclease, rad2, were all essential. Targeted genes were classified as non-essential if knockouts were obtained and essential based on statistical analysis and/or by demonstrating the inability to isolate chromosomal knockouts except in the presence of a complementing plasmid copy of the gene. Conclusion The results showed that ten

  3. Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins

    Directory of Open Access Journals (Sweden)

    Whittington Jessica

    2007-07-01

    Full Text Available Abstract Background The secretion time course of Bacillus anthracis strain RA3R (pXO1+/pXO2- during early, mid, and late log phase were investigated under conditions that simulate those encountered in the host. All of the identified proteins were analyzed by different software algorithms to characterize their predicted mode of secretion and cellular localization. In addition, immunogenic proteins were identified using sera from humans with cutaneous anthrax. Results A total of 275 extracellular proteins were identified by a combination of LC MS/MS and MALDI-TOF MS. All of the identified proteins were analyzed by SignalP, SecretomeP, PSORT, LipoP, TMHMM, and PROSITE to characterize their predicted mode of secretion, cellular localization, and protein domains. Fifty-three proteins were predicted by SignalP to harbor the cleavable N-terminal signal peptides and were therefore secreted via the classical Sec pathway. Twenty-three proteins were predicted by SecretomeP for secretion by the alternative Sec pathway characterized by the lack of typical export signal. In contrast to SignalP and SecretomeP predictions, PSORT predicted 171 extracellular proteins, 7 cell wall-associated proteins, and 6 cytoplasmic proteins. Moreover, 51 proteins were predicted by LipoP to contain putative Sec signal peptides (38 have SpI sites, lipoprotein signal peptides (13 have SpII sites, and N-terminal membrane helices (9 have transmembrane helices. The TMHMM algorithm predicted 25 membrane-associated proteins with one to ten transmembrane helices. Immunogenic proteins were also identified using sera from patients who have recovered from anthrax. The charge variants (83 and 63 kDa of protective antigen (PA were the most immunodominant secreted antigens, followed by charge variants of enolase and transketolase. Conclusion This is the first description of the time course of protein secretion for the pathogen Bacillus anthracis. Time course studies of protein secretion and

  4. Pathology of wild-type and toxin-independent Bacillus anthracis meningitis in rabbits.

    Directory of Open Access Journals (Sweden)

    Assa Sittner

    Full Text Available Hemorrhagic meningitis is considered a complication of anthrax and was reported in about 50% of deadly cases in humans and non-human primates (NHP. Recently we demonstrated in Guinea pigs and rabbits that 100% of the B. anthracis-infected animals presented histopathology of meningitis at the time of death, some without any sign of hemorrhage. A similar pathology was observed in animals that succumbed following infection with the toxin deficient mutant, thus indicating that anthrax meningitis is a toxin-independent phenomenon. In this manuscript we describe a histopathological study of the B. anthracis infection of the central nervous system (CNS. Though we could find sporadic growth of the bacteria around blood vessels in the cortex, we report that the main infiltration route is the choroid plexus. We found massive destruction of entire sections of the choroid plexus coupled with massive aggregation of bacilli in the ventricles, in close proximity to the parenchyma. The choroid plexus also contained significant amounts of intravascular bacterial aggregates, often enclosed in what appear to be fibrin-like clots. The high concentration of these aggregates in areas of significant tissue destruction combined with the fact that capsular B. anthracis bacteria have a low tendency to adhere to endothelial cells, might suggest that these clots are used as an adherence mechanism by the bacteria. The major histopathological finding is meningitis. We find massive bacterial growth in the meninges without evidence of encephalitis, even when the bacteria emerge from a parenchymal blood vessel. Erythrocytes were present within the meningeal space but no clear vasculitis could be detected. Histology of the brain stem indicates meningitis, edema and hemorrhages that might explain death from suffocation due to direct damage to the respiratory center. All of these processes are toxin-independent, since they were observed following infection with either the wild

  5. The genome of a Bacillus isolate causing anthrax in chimpanzees combines chromosomal properties of B. cereus with B. anthracis virulence plasmids.

    Directory of Open Access Journals (Sweden)

    Silke R Klee

    Full Text Available Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as "B. cereus variety (var. anthracis".

  6. Physical Sequestration of Bacillus anthracis in the Pulmonary Capillaries in Terminal Infection.

    Science.gov (United States)

    Jouvion, Gregory; Corre, Jean-Philippe; Khun, Huot; Moya-Nilges, Marie; Roux, Pascal; Latroche, Claire; Tournier, Jean-Nicolas; Huerre, Michel; Chrétien, Fabrice; Goossens, Pierre L

    2016-07-15

    The lung is the terminal target of Bacillus anthracis before death, whatever the route of infection (cutaneous, inhalational, or digestive). During a cutaneous infection in absence of toxins, we observed encapsulated bacteria colonizing the alveolar capillary network, bacteria and hemorrhages in alveolar and bronchiolar spaces, and hypoxic foci in the lung (endothelial cells) and brain (neurons and neuropil). Circulating encapsulated bacteria were as chains of approximately 13 µm in length. Bacteria of such size were immediately trapped within the lung capillary network, but bacteria of shorter length were not. Controlling lung-targeted pathology would be beneficial for anthrax treatment. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  7. Esterase activity as a novel parameter of spore germination in Bacillus anthracis

    International Nuclear Information System (INIS)

    Ferencko, Linda; Cote, Mindy A.; Rotman, Boris

    2004-01-01

    Spores of Bacillus anthracis were shown to produce esterase activity about 4 min after exposure to conventional germinants such as combinations of amino acids and purine ribosides. Neither amino acids nor ribosides alone induce germination and esterase activity. Expression of esterase activity was chloramphenicol resistant, and correlated with loss of spore refractivity, a traditional parameter of early germination. Based on these observations, we hypothesized that esterase activity could be used as a novel parameter for quantifying early events during spore germination. To test this hypothesis, we measured expression of esterase activity under a variety of germinating conditions. Using diacetyl fluorescein as fluorogenic substrate of esterases, we demonstrated that esterase activity was invariably induced whenever spores were triggered by known germinants. Moreover, D-alanine, an inhibitor of L-alanine-mediated germination, was found to significantly inhibit expression of esterase activity. In terms of molecular mechanisms, esterase expression could represent activation of proteases at the onset of spore germination

  8. Proteomic signatures differentiating Bacillus anthracis Sterne sporulation on soil relative to laboratory media.

    Science.gov (United States)

    Wunschel, D S; Hutchison, J R; Deatherage Kaiser, B L; Merkley, E D; Hess, B M; Lin, A; Warner, M G

    2017-12-18

    The process of sporulation is vital for the stability and infectious cycle of Bacillus anthracis. The spore is the infectious form of the organism and therefore relevant to biodefense. While the morphological and molecular events occurring during sporulation have been well studied, the influence of growth medium and temperature on the proteins expressed in sporulated cultures is not well understood. Understanding the features of B. anthracis sporulation specific to natural vs. laboratory production will address an important question in microbial forensics. In an effort to bridge this knowledge gap, a system for sporulation on two types of agar-immobilized soils was used for comparison to cultures sporulated on two common types of solid laboratory media, and one liquid sporulation medium. The total number of proteins identified as well as their identity differed between samples generated in each medium and growth temperature, demonstrating that sporulation environment significantly impacts the protein content of the spore. In addition, a subset of proteins common in all of the soil-cultivated samples was distinct from the expression profiles in laboratory medium (and vice versa). These differences included proteins involved in thiamine and phosphate metabolism in the sporulated cultures produced on soils with a notable increase in expression of ATP binding cassette (ABC) transporters annotated to be for phosphate and antimicrobial peptides. A distinct set of ABC transporters for amino acids, sugars and oligopeptides were found in cultures produced on laboratory media as well as increases in carbon and amino acid metabolism-related proteins. These protein expression changes indicate that the sporulation environment impacts the protein profiles in specific ways that are reflected in the metabolic and membrane transporter proteins present in sporulated cultures.

  9. Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu

    Energy Technology Data Exchange (ETDEWEB)

    Schnicker, Nicholas J. [Department; Razzaghi, Mortezaali [Department; Guha Thakurta, Sanjukta [Department; Chakravarthy, Srinivas [Biophysics; Dey, Mishtu [Department

    2017-10-17

    Prolyl hydroxylation is a very common post-translational modification and plays many roles in eukaryotes such as collagen stabilization, hypoxia sensing, and controlling protein transcription and translation. There is a growing body of evidence that suggests that prokaryotes contain prolyl 4-hydroxylases (P4Hs) homologous to the hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes that act on elongation factor Tu (EFTu) and are likely involved in the regulation of bacterial translation. Recent biochemical and structural studies with a PHD from Pseudomonas putida (PPHD) determined that it forms a complex with EFTu and hydroxylates a prolyl residue of EFTu. Moreover, while animal, plant, and viral P4Hs act on peptidyl proline, most prokaryotic P4Hs have been known to target free l-proline; the exceptions include PPHD and a P4H from Bacillus anthracis (BaP4H) that modifies collagen-like proline-rich peptides. Here we use biophysical and mass spectrometric methods to demonstrate that BaP4H recognizes full-length BaEFTu and a BaEFTu 9-mer peptide for site-specific proline hydroxylation. Using size-exclusion chromatography coupled small-angle X-ray scattering (SEC–SAXS) and binding studies, we determined that BaP4H forms a 1:1 heterodimeric complex with BaEFTu. The SEC–SAXS studies reveal dissociation of BaP4H dimeric subunits upon interaction with BaEFTu. While BaP4H is unusual within bacteria in that it is structurally and functionally similar to the animal PHDs and collagen P4Hs, respectively, this work provides further evidence of its promiscuous substrate recognition. It is possible that the enzyme might have evolved to hydroxylate a universally conserved protein in prokaryotes, similar to the PHDs, and implies a functional role in B. anthracis.

  10. Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

    Science.gov (United States)

    Terwilliger, Austen; Swick, Michelle C.; Pflughoeft, Kathryn J.; Pomerantsev, Andrei; Lyons, C. Rick; Koehler, Theresa M.

    2015-01-01

    ABSTRACT Bacteria sustain an infection by acquiring nutrients from the host to support replication. The host sequesters these nutrients as a growth-restricting strategy, a concept termed “nutritional immunity.” Historically, the study of nutritional immunity has centered on iron uptake because many bacteria target hemoglobin, an abundant circulating protein, as an iron source. Left unresolved are the mechanisms that bacteria use to attain other nutrients from host sources, including amino acids. We employed a novel medium designed to mimic the chemical composition of human serum, and we show here that Bacillus anthracis, the causative agent of anthrax disease, proteolyzes human hemoglobin to liberate essential amino acids which enhance its growth. This property can be traced to the actions of InhA1, a secreted metalloprotease, and extends to at least three other serum proteins, including serum albumin. The results suggest that we must also consider proteolysis of key host proteins to be a way for bacterial pathogens to attain essential nutrients, and we provide an experimental framework to determine the host and bacterial factors involved in this process. IMPORTANCE The mechanisms by which bacterial pathogens acquire nutrients during infection are poorly understood. Here we used a novel defined medium that approximates the chemical composition of human blood serum, blood serum mimic (BSM), to better model the nutritional environment that pathogens encounter during bacteremia. Removing essential amino acids from BSM revealed that two of the most abundant proteins in blood—hemoglobin and serum albumin—can satiate the amino acid requirement for Bacillus anthracis, the causative agent of anthrax. We further demonstrate that hemoglobin is proteolyzed by the secreted protease InhA1. These studies highlight that common blood proteins can be a nutrient source for bacteria. They also challenge the historical view that hemoglobin is solely an iron source for

  11. Guardians of the mycobacterial genome: A review on DNA repair systems in Mycobacterium tuberculosis.

    Science.gov (United States)

    Singh, Amandeep

    2017-12-01

    The genomic integrity of Mycobacterium tuberculosis is continuously threatened by the harsh survival conditions inside host macrophages, due to immune and antibiotic stresses. Faithful genome maintenance and repair must be accomplished under stress for the bacillus to survive in the host, necessitating a robust DNA repair system. The importance of DNA repair systems in pathogenesis is well established. Previous examination of the M. tuberculosis genome revealed homologues of almost all the major DNA repair systems, i.e. nucleotide excision repair (NER), base excision repair (BER), homologous recombination (HR) and non-homologous end joining (NHEJ). However, recent developments in the field have pointed to the presence of novel proteins and pathways in mycobacteria. Homologues of archeal mismatch repair proteins were recently reported in mycobacteria, a pathway previously thought to be absent. RecBCD, the major nuclease-helicase enzymes involved in HR in E. coli, were implicated in the single-strand annealing (SSA) pathway. Novel roles of archeo-eukaryotic primase (AEP) polymerases, previously thought to be exclusive to NHEJ, have been reported in BER. Many new proteins with a probable role in DNA repair have also been discovered. It is now realized that the DNA repair systems in M. tuberculosis are highly evolved and have redundant backup mechanisms to mend the damage. This review is an attempt to summarize our current understanding of the DNA repair systems in M. tuberculosis.

  12. Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase.

    Science.gov (United States)

    Brissett, Nigel C; Martin, Maria J; Pitcher, Robert S; Bianchi, Julie; Juarez, Raquel; Green, Andrew J; Fox, Gavin C; Blanco, Luis; Doherty, Aidan J

    2011-01-21

    In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a polymerase:DNA complex in a preternary intermediate state. This polymerase complex occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension of short incoming termini. We also demonstrate that the invariant Arg(220), contained in a conserved loop (loop 2), plays an essential role in catalysis by regulating binding of a second metal ion in the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or noncomplementary DNA ends, thus promoting break repair and minimizing sequence loss during DSB repair. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. [Efficacy of enterocin S760 in treatment of mice with anthrax infection due to Bacillus anthracis M-71].

    Science.gov (United States)

    Svetoch, E A; Borzilov, A I; Eruslanov, B V; Korobova, O V; Kombarova, T I; Levchuk, V P; Teĭmurazov, M G; Stepanshin, Iu G; Marinin, L I; Diatlov, I A

    2011-01-01

    The therapeutic efficacy of enterocin S760, a broad spectrum antimicrobial peptide produced by Enterococcus faecium LWP760 was tested on mice infected with Bacillus anthracis M-71 to induce anthrax (second Tsenkovsky's vaccine). Intraperitoneal four-, two- or one-fold administration of the peptide in a dose of 25 mg/kg for 10 days for prophylactic (1 hour after the contamination) and therapeutic (24 hours after the contamination) purposes prevented or cured the infection in 90-100% of the mice versus the 100-percent lethality in the control (untreated animals). The antimicrobial activity of enterocin S760 against B. anthracis M-71 in vivo correlated with activity in vitro. Enterocin S760 is considered a novel promising antimicrobial for the treatment of grampositive and gramnegative infections.

  14. Rapid Antimicrobial Susceptibility Testing of Bacillus anthracis, Yersinia pestis, and Burkholderia pseudomallei by Use of Laser Light Scattering Technology.

    Science.gov (United States)

    Bugrysheva, Julia V; Lascols, Christine; Sue, David; Weigel, Linda M

    2016-06-01

    Rapid methods to determine antimicrobial susceptibility would assist in the timely distribution of effective treatment or postexposure prophylaxis in the aftermath of the release of bacterial biothreat agents such as Bacillus anthracis, Yersinia pestis, or Burkholderia pseudomallei Conventional susceptibility tests require 16 to 48 h of incubation, depending on the bacterial species. We evaluated a method that is based on laser light scattering technology that measures cell density in real time. We determined that it has the ability to rapidly differentiate between growth (resistant) and no growth (susceptible) of several bacterial threat agents in the presence of clinically relevant antimicrobials. Results were available in 10 h of incubation. Use of laser scattering technology decreased the time required to determine antimicrobial susceptibility by 50% to 75% for B. anthracis, Y. pestis, and B. pseudomallei compared to conventional methods. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  15. Detection of Bacillus anthracis spores by super-paramagnetic lateral-flow immunoassays based on "Road Closure".

    Science.gov (United States)

    Wang, Dian-Bing; Tian, Bo; Zhang, Zhi-Ping; Wang, Xu-Ying; Fleming, Joy; Bi, Li-Jun; Yang, Rui-Fu; Zhang, Xian-En

    2015-05-15

    Detection of Bacillus anthracis in the field, whether as a natural infection or as a biothreat remains challenging. Here we have developed a new lateral-flow immunochromatographic assay (LFIA) for B. anthracis spore detection based on the fact that conjugates of B. anthracis spores and super-paramagnetic particles labeled with antibodies will block the pores of chromatographic strips and form retention lines on the strips, instead of the conventionally reported test lines and control lines in classic LFIA. As a result, this new LFIA can simultaneously realize optical, magnetic and naked-eye detection by analyzing signals from the retention lines. As few as 500-700 pure B. anthracis spores can be recognized with CV values less than 8.31% within 5 min of chromatography and a total time of 20 min. For powdery sample tests, this LFIA can endure interference from 25% (w/v) milk, 10% (w/v) baking soda and 10% (w/v) starch without any sample pre-treatment, and has a corresponding detection limit of 6×10(4) spores/g milk powder, 2×10(5) spores/g starch and 5×10(5) spores/g baking soda. Compared with existing methods, this new approach is very competitive in terms of sensitivity, specificity, cost and ease of operation. This proof-of-concept study can also be extended for detection of many other large-sized analytes. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Inhibiting Inosine Hydrolase and Alanine Racemase to Enhance the Germination of Bacillus anthracis Sterne Spores: Potential Spore Decontamination Strategies

    Science.gov (United States)

    2015-06-19

    decontamination strategies>> Maryline DEFEZ 1𔃼, Melissa HUNTER3J Susan WELKOS :~J Christopher COTE3 1 University Grenoble-Alpes, Grenoble, France. 1...inosine hydrolase and alanine racemase to enhance the germination of Bacillus anthracis Sterne spores potential spore decontamination strategies 5a...8217 • Accidentally in Humans • Natural reservoir is soil • Anthrax Disease Cycle: - animals infected by soilborne spores in food and water or bites from certain

  17. In Vitro and In Vivo Activity of Omadacycline Against Two Biothreat Pathogens: Bacillus Anthracis and Yersinia Pestis

    Science.gov (United States)

    2013-02-27

    visually at 18-24 hours (B. anthracis) or 42-48 hours (Y. pestis) and also by absorbance at 600 nm (SpectroMax M2, Molecular Devices). Thirty...certain uncomplicated infections; warns about disabling side effects that can occur together. May 12, 2016. Accessed August 29, 2016. Flamm RK, Rhomberg... odontology in the management of bioterrorism. In Evidence-Based Forensic Dentistry. Springer-Verlag Berlin Heidelberg 2013. pp. 149-152. Rotz LD

  18. Immunoassay for Capsular Antigen of Bacillus anthracis Enables Rapid Diagnosis in a Rabbit Model of Inhalational Anthrax.

    Directory of Open Access Journals (Sweden)

    Marcellene A Gates-Hollingsworth

    Full Text Available Inhalational anthrax is a serious biothreat. Effective antibiotic treatment of inhalational anthrax requires early diagnosis; the further the disease has progressed, the less the likelihood for cure. Current means for diagnosis such as blood culture require several days to a result and require advanced laboratory infrastructure. An alternative approach to diagnosis is detection of a Bacillus anthracis antigen that is shed into blood and can be detected by rapid immunoassay. The goal of the study was to evaluate detection of poly-γ-D-glutamic acid (PGA, the capsular antigen of B. anthracis, as a biomarker surrogate for blood culture in a rabbit model of inhalational anthrax. The mean time to a positive blood culture was 26 ± 5.7 h (mean ± standard deviation, whereas the mean time to a positive ELISA was 22 ± 4.2 h; P = 0.005 in comparison with blood culture. A lateral flow immunoassay was constructed for detection of PGA in plasma at concentrations of less than 1 ng PGA/ml. Use of the lateral flow immunoassay for detection of PGA in the rabbit model found that antigen was detected somewhat earlier than the earliest time point at which the blood culture became positive. The low cost, ease of use, and rapid time to result of the lateral flow immunoassay format make an immunoassay for PGA a viable surrogate for blood culture for detection of infection in individuals who have a likelihood of exposure to B. anthracis.

  19. Cloning, expression, and characterization of recombinant nitric oxide synthase-like protein from Bacillus anthracis

    International Nuclear Information System (INIS)

    Midha, Shuchi; Mishra, Rajeev; Aziz, M.A.; Sharma, Meenakshi; Mishra, Ashish; Khandelwal, Puneet; Bhatnagar, Rakesh

    2005-01-01

    Nitric oxide synthase (NOS) is amongst a family of evolutionarily conserved enzymes, involved in a multi-turnover process that results in NO as a product. The significant role of NO in various pathological and physiological processes has created an interest in this enzyme from several perspectives. This study describes for the first time, cloning and expression of a NOS-like protein, baNOS, from Bacillus anthracis, a pathogenic bacterium responsible for causing anthrax. baNOS was expressed in Escherichia coli as a soluble and catalytically active enzyme. Homology models generated for baNOS indicated that the key structural features that are involved in the substrate and active site interaction have been highly conserved. Further, the behavior of baNOS in terms of heme-substrate interactions and heme-transitions was studied in detail. The optical perturbation spectra of the heme domain demonstrated that the ligands perturb the heme site in a ligand specific manner. baNOS forms a five-coordinate, high-spin complex with L-arginine analogs and a six-coordinate low-spin complex with inhibitor imidazole. Studies indicated that the binding of L-arginine, N ω -hydroxy-L-arginine, and imidazole produces various spectroscopic species that closely correspond to the equivalent complexes of mammalian NOS. The values of spectral binding constants further corroborated these results. The overall conservation of the key structural features and the correlation of heme-substrate interactions in baNOS and mammalian NOS, thus, point towards an interesting phenomenon of convergent evolution. Importantly, the NO generated by NOS of mammalian macrophages plays a potent role in antimicrobicidal activity. Because of the existence of high structural and behavioral similarity between mammalian NOS and baNOS, we propose that NO produced by B. anthracis may also have a pivotal pathophysiological role in anthrax infection. Therefore, this first report of characterization of a NOS-like protein

  20. Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture.

    Science.gov (United States)

    Hutchison, J R; Piepel, G F; Amidan, B G; Hess, B M; Sydor, M A; Deatherage Kaiser, B L

    2018-05-01

    We evaluated the effects of Bacillus anthracis surrogates, low surface concentrations, surface materials and assay methods on false-negative rate (FNR) and limit of detection (LOD 95 ) for recovering Bacillus spores using a macrofoam-swab sampling procedure. Bacillus anthracis Sterne or Bacillus atrophaeus Nakamura spores were deposited over a range of low target concentrations (2-500 per coupon) onto glass, stainless steel, vinyl tile and plastic. Samples were assayed using a modified Rapid Viability-PCR (mRV-PCR) method and the traditional plate culture method to obtain FNR and LOD 95 results. Mean FNRs tended to be lower for mRV-PCR compared to culturing, and increased as spore concentration decreased for all surface materials. Surface material, but not B. anthracis surrogate, influenced FNRs with the mRV-PCR method. The mRV-PCR LOD 95 was lowest for glass and highest for vinyl tile. LOD 95 values overall were lower for mRV-PCR than for the culture method. This study adds to the limited data on FNR and LOD 95 for mRV-PCR and culturing methods with low concentrations of B. anthracis sampled from various surface materials by the CDC macrofoam-swab method. These are key inputs for planning characterization and clearance studies for low contamination levels of B. anthracis. © 2018 The Society for Applied Microbiology.

  1. Ultrasensitive DNA sequence detection using nanoscale ZnO sensor arrays

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Nitin; Dorfman, Adam; Hahm, Jong-in [Department of Chemical Engineering, Pennsylvania State University, 160 Fenske Laboratory, University Park, PA 16802 (United States)

    2006-06-28

    We report that engineered nanoscale zinc oxide structures can be effectively used for the identification of the biothreat agent, Bacillus anthracis by successfully discriminating its DNA sequence from other genetically related species. We explore both covalent and non-covalent linking schemes in order to couple probe DNA strands to the zinc oxide nanostructures. Hybridization reactions are performed with various concentrations of target DNA strands whose sequence is unique to Bacillus anthracis. The use of zinc oxide nanomaterials greatly enhances the fluorescence signal collected after carrying out duplex formation reaction. Specifically, the covalent strategy allows detection of the target species at sample concentrations at a level as low as a few femtomolar as compared to the detection sensitivity in the tens of nanomolar range when using the non-covalent scheme. The presence of the underlying zinc oxide nanomaterials is critical in achieving increased fluorescence detection of hybridized DNA and, therefore, accomplishing rapid and extremely sensitive identification of the biothreat agent. We also demonstrate the easy integration potential of nanoscale zinc oxide into high density arrays by using various types of zinc oxide sensor prototypes in the DNA sequence detection. When combined with conventional automatic sample handling apparatus and computerized fluorescence detection equipment, our approach can greatly promote the use of zinc oxide nanomaterials as signal enhancing platforms for rapid, multiplexed, high-throughput, highly sensitive, DNA sensor arrays.

  2. Ultrasensitive DNA sequence detection using nanoscale ZnO sensor arrays

    International Nuclear Information System (INIS)

    Kumar, Nitin; Dorfman, Adam; Hahm, Jong-in

    2006-01-01

    We report that engineered nanoscale zinc oxide structures can be effectively used for the identification of the biothreat agent, Bacillus anthracis by successfully discriminating its DNA sequence from other genetically related species. We explore both covalent and non-covalent linking schemes in order to couple probe DNA strands to the zinc oxide nanostructures. Hybridization reactions are performed with various concentrations of target DNA strands whose sequence is unique to Bacillus anthracis. The use of zinc oxide nanomaterials greatly enhances the fluorescence signal collected after carrying out duplex formation reaction. Specifically, the covalent strategy allows detection of the target species at sample concentrations at a level as low as a few femtomolar as compared to the detection sensitivity in the tens of nanomolar range when using the non-covalent scheme. The presence of the underlying zinc oxide nanomaterials is critical in achieving increased fluorescence detection of hybridized DNA and, therefore, accomplishing rapid and extremely sensitive identification of the biothreat agent. We also demonstrate the easy integration potential of nanoscale zinc oxide into high density arrays by using various types of zinc oxide sensor prototypes in the DNA sequence detection. When combined with conventional automatic sample handling apparatus and computerized fluorescence detection equipment, our approach can greatly promote the use of zinc oxide nanomaterials as signal enhancing platforms for rapid, multiplexed, high-throughput, highly sensitive, DNA sensor arrays

  3. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  4. Regulation of expression of a select group of Bacillus anthracis spore coat proteins.

    Science.gov (United States)

    Aronson, Arthur

    2018-04-01

    The spore coat of Bacilli is a relatively complex structure comprised of about 70 species of proteins in 2 or 3 layers. While some are involved in assembly or protection, the regulation of many are not well defined so lacZ transcriptional fusions were constructed to six Bacillus anthracis spore coat genes in order to gain insight into their possible functions. The genes were selected on the basis of the location of the encoded proteins within the coat and distribution among spore forming species. Conditions tested were temperature and media either as solid or liquid. The most extensive differences were for the relatively well expressed fusions to the cotH and cotM genes, which were greatest at 30°C on plates of a nutrient rich medium. The cotJ operon was moderately expressed under all conditions although somewhat higher on enriched plates at 30°C. Cot S was low under all conditions except for a substantial increase in biofilm medium. Cot∝ and cotF were essentially invariant with a somewhat greater expression in the more enriched medium. The capacity of a subset of coat genes to respond to various conditions reflects a flexibility in spore coat structure that may be necessary for adaptation to environmental challenges. This could account, at least in part, for the complexity of this structure.

  5. The regulated synthesis of a Bacillus anthracis spore coat protein that affects spore surface properties.

    Science.gov (United States)

    Aronson, A; Goodman, B; Smith, Z

    2014-05-01

    Examine the regulation of a spore coat protein and the effects on spore properties. A c. 23 kDa band in coat/exosporial extracts of Bacillus anthracis Sterne spores varied in amount depending upon the conditions of sporulation. It was identified by MALDI as a likely orthologue of ExsB of Bacillus cereus. Little if any was present in an exosporial preparation with a location to the inner coat/cortex region established by spore fractionation and immunogold labelling of electron micrograph sections. Because of its predominant location in the inner coat, it has been renamed Cotγ. It was relatively deficient in spores produced at 37°C and when acidic fermentation products were produced a difference attributable to transcriptional regulation. The deficiency or absence of Cotγ resulted in a less robust exosporium positioned more closely to the coat. These spores were less hydrophobic and germinated somewhat more rapidly. Hydrophobicity and appearance were rescued in the deletion strain by introduction of the cotγ gene. The deficiency or lack of a protein largely found in the inner coat altered spore hydrophobicity and surface appearance. The regulated synthesis of Cotγ may be a paradigm for other spore coat proteins with unknown functions that modulate spore properties in response to environmental conditions. © 2014 The Society for Applied Microbiology.

  6. The Saccharomyces boulardii CNCM I-745 Strain Shows Protective Effects against the B. anthracis LT Toxin

    Directory of Open Access Journals (Sweden)

    Rodolphe Pontier-Bres

    2015-10-01

    Full Text Available The probiotic yeast Saccharomyces boulardii (S. boulardii has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax.

  7. Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells.

    Directory of Open Access Journals (Sweden)

    Scott A Walper

    Full Text Available Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.

  8. Effect of animal sera on Bacillus anthracis Sterne spore germination and vegetative cell growth.

    Science.gov (United States)

    Bensman, M D; Mackie, R S; Minter, Z A; Gutting, B W

    2012-08-01

     The aims of this work were to investigate the effects of sera on B. anthracis Sterne germination and growth. Sera examined included human, monkey and rabbit sera, as well as sera from eight other species.  Standard dilution plate assay (with and without heat kill) was used as a measure of germination, and spectroscopy was used to measure growth. In addition, a Coulter Counter particle counter was used to monitor germination and growth based on bacterial size. Spores germinated best in foetal bovine and monkey sera, moderately with human sera and showed limited germination in the presence of rabbit or rat sera. Vegetative bacteria grew best in foetal bovine sera and moderately in rabbit sera. Human and monkey sera supported little growth of vegetative bacteria.  The data suggested sera can have a significant impact on germination and growth of Sterne bacteria.  These data should be considered when conducting in vitro cell culture studies and may aid in interpreting in vivo infection studies. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

  9. Intranasal immunization with protective antigen of Bacillus anthracis induces a long-term immunological memory response.

    Science.gov (United States)

    Woo, Sun-Je; Kang, Seok-Seong; Park, Sung-Moo; Yang, Jae Seung; Song, Man Ki; Yun, Cheol-Heui; Han, Seung Hyun

    2015-10-01

    Although intranasal vaccination has been shown to be effective for the protection against inhalational anthrax, establishment of long-term immunity has yet to be achieved. Here, we investigated whether intranasal immunization with recombinant protective antigen (rPA) of Bacillus anthracis induces immunological memory responses in the mucosal and systemic compartments. Intranasal immunization with rPA plus cholera toxin (CT) sustained PA-specific antibody responses for 6 months in lung, nasal washes, and vaginal washes as well as serum. A significant induction of PA-specific memory B cells was observed in spleen, cervical lymph nodes (CLNs) and lung after booster immunization. Furthermore, intranasal immunization with rPA plus CT remarkably generated effector memory CD4(+) T cells in the lung. PA-specific CD4(+) T cells preferentially increased the expression of Th1- and Th17-type cytokines in lung, but not in spleen or CLNs. Collectively, the intranasal immunization with rPA plus CT promoted immunologic memory responses in the mucosal and systemic compartments, providing long-term immunity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Bacillus anthracis Co-Opts Nitric Oxide and Host Serum Albumin for Pathogenicity in Hypoxic Conditions

    Directory of Open Access Journals (Sweden)

    Stephen eSt John

    2013-05-01

    Full Text Available Bacillus anthracis is a dangerous pathogen of humans and many animal species. Its virulence has been mainly attributed to the production of Lethal and Edema toxins as well as the antiphagocytic capsule. Recent data indicate that the nitric oxide (NO synthase (baNOS plays an important pathogenic role at the early stage of disease by protecting bacteria from the host reactive species and S-nytrosylating the mitochondrial proteins in macrophages. In this study we for the first time present evidence that bacteria-derived NO participates in the generation of highly reactive oxidizing species which could be abolished by the NOS inhibitor L-NAME, free thiols, and superoxide dismutase but not catalase. The formation of toxicants is likely a result of the simultaneous formation of NO and superoxide leading to a labile peroxynitrite and its stable decomposition product, nitrogen dioxide. The toxicity of bacteria could be potentiated in the presence of bovine serum albumin. This effect is consistent with the property of serum albumin to serves as a trap of a volatile NO accelerating its reactions. Our data suggest that during infection in the hypoxic environment of pre-mortal host the accumulated NO is expected to have a broad toxic impact on host cell functions.

  11. Measurements of the Ultraviolet Fluorescence Cross Sections and Spectra of Bacillus Anthracis Simulants

    Energy Technology Data Exchange (ETDEWEB)

    Stephens, J.R.

    1998-09-01

    Measurements of the ultraviolet autofluorescence spectra and absolute cross sections of the Bacillus anthracis (Ba) simulants Bacillus globigii (Bg), Bacillus megaterium (Bm), Bacillus subtilis (Bs), and Bacillus cereus (Bc) were measured. Fluorescence spectra and cross sections of pine pollen (Pina echinata) were measured for comparison. Both dried vegetative cells and spores separated from the sporulated vegetative material were studied. The spectra were obtained by suspending a small number (<10) of particles in air in our Single Particle Spectroscopy Apparatus (SPSA), illuminating the particles with light from a spectrally filtered arc lamp, and measuring the fluorescence spectra of the particles. The illumination was 280 nm (20 nm FWHM) and the fluorescence spectra was measured between 300 and 450 nm. The fluorescence cross section of vegetative Bg peaks at 320 nm with a maximum cross section of 5 X 10{sup -14} cm{sup 2}/sr-nm-particle while the Bg spore fluorescence peaks at 310 nm with peak fluorescence of 8 X 10{sup -15} cm{sup 2}/sr-nm-particle. Pine pollen particles showed a higher fluorescence peaking at 355 nm with a cross section of 1.7 X 10{sup -13} cm{sup 2}/sr-nm-particle. Integrated cross sections ranged from 3.0 X 10{sup -13} for the Bg spores through 2.25 X 10{sup -12} (cm{sup 2}/sr-particle) for the vegetative cells.

  12. The Saccharomyces boulardii CNCM I-745 strain shows protective effects against the B. anthracis LT toxin.

    Science.gov (United States)

    Pontier-Bres, Rodolphe; Rampal, Patrick; Peyron, Jean-François; Munro, Patrick; Lemichez, Emmanuel; Czerucka, Dorota

    2015-10-30

    The probiotic yeast Saccharomyces boulardii (S. boulardii) has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT) of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax.

  13. First detection of Bacillus anthracis in feces of free-ranging raptors from central Argentina.

    Science.gov (United States)

    Saggese, Miguel D; Noseda, Ramón P; Uhart, Marcela M; Deem, Sharon L; Ferreyra, Hebe; Romano, Marcelo C; Ferreyra-Armas, María C; Hugh-Jones, Martin

    2007-01-01

    Prevalence of anthrax spores in feces of raptors was determined from samples collected in November-December 2000 and April-May 2001 in an agricultural region of Santa Fé province, Argentina. Feces were tested from 48 birds of six raptor species. One of 14 chimango caracaras (Milvago chimango) and one of eight road-side hawks (Buteo magnirostris) tested positive. The prevalence of Bacillus anthracis spores in feces for the six species was 4% (n=48). The prevalence was 7% (n=14) for chimango caracaras, 13% for road-side hawks (n=8), and 0% for the remaining species (Burrowing owl [Speotyto cunicularia] [n=17], Swainson's hawk [Buteo swainsoni] [n=3], Aplomado falcon [Falco femoralis] [n=2], and American kestrel [Falco sparverius] [n=4]). Grouped by their feeding habits, prevalence for scavenger species was not significantly different than for predators (7% vs. 3%, P>0.999). This study provides evidence that in central Argentina scavenger and non-scavenger raptors may have a role in the epidemiology of anthrax. Long-term studies to determine the extent of this potential involvement in the epidemiology of anthrax in central Argentina are required.

  14. Domain structure of a NHEJ DNA repair ligase from Mycobacterium tuberculosis.

    Science.gov (United States)

    Pitcher, Robert S; Tonkin, Louise M; Green, Andrew J; Doherty, Aidan J

    2005-08-19

    A prokaryotic non-homologous end-joining (NHEJ) system for the repair of DNA double-strand breaks (DSBs), composed of a Ku homodimer (Mt-Ku) and a multidomain multifunctional ATP-dependent DNA ligase (Mt-Lig), has been described recently in Mycobacterium tuberculosis. Mt-Lig exhibits polymerase and nuclease activity in addition to DNA ligation activity. These functions were ascribed to putative polymerase, nuclease and ligase domains that together constitute a monomeric protein. Here, the separate polymerase, nuclease and ligase domains of Mt-Lig were cloned individually, over-expressed and the soluble proteins purified to homogeneity. The polymerase domain demonstrated DNA-dependent RNA primase activity, catalysing the synthesis of unprimed oligoribonucleotides on single-stranded DNA templates. The polymerase domain can also extend DNA in a template-dependent manner. This activity was eliminated when the catalytic aspartate residues were replaced with alanine. The ligase domain catalysed the sealing of nicked double-stranded DNA designed to mimic a DSB, consistent with the role of Mt-Lig in NHEJ. Deletion of the active-site lysine residue prevented the formation of an adenylated ligase complex and consequently thwarted ligation. The nuclease domain did not function independently as a 3'-5' exonuclease. DNA-binding assays revealed that both the polymerase and ligase domains bind DNA in vitro, the latter with considerably higher affinity. Mt-Ku directly stimulated the polymerase and nuclease activities of Mt-Lig. The polymerase domain bound Mt-Ku in vitro, suggesting it may recruit Mt-Lig to Ku-bound DNA in vivo. Consistent with these data, Mt-Ku stimulated the primer extension activity of the polymerase domain, suggestive of a functional interaction relevant to NHEJ-mediated DSB repair processes.

  15. Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing.

    Science.gov (United States)

    Senkevich, Tatiana G; Bruno, Daniel; Martens, Craig; Porcella, Stephen F; Wolf, Yuri I; Moss, Bernard

    2015-09-01

    Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.

  16. Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Rosemary S Turingan

    Full Text Available BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed "Rapid Focused Sequencing," allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental

  17. Development and field testing of a mobile chlorine dioxide generation system for the decontamination of buildings contaminated with Bacillus anthracis

    International Nuclear Information System (INIS)

    Wood, Joseph P.; Blair Martin, G.

    2009-01-01

    The numerous buildings that became contaminated with Bacillus anthracis (the bacterium causing the disease anthrax) in 2001, and more recent B. anthracis - related events, point to the need to have effective decontamination technologies for buildings contaminated with biological threat agents. The U.S. Government developed a portable chlorine dioxide (ClO 2 ) generation system to decontaminate buildings contaminated with B. anthracis spores, and this so-called mobile decontamination trailer (MDT) prototype was tested through a series of three field trials. The first test of the MDT was conducted at Fort McClellan in Anniston, AL. during October 2004. Four test attempts occurred over two weekends; however, a number of system problems resulted in termination of the activity prior to any ClO 2 introduction into the test building. After making several design enhancements and equipment changes, the MDT was subjected to a second test. During this test, extensive leak checks were made using argon and nitrogen in lieu of chlorine gas; each subsystem was checked for functionality, and the MDT was operated for 24 h. This second test demonstrated the MDT flow and control systems functioned satisfactorily, and thus it was decided to proceed to a third, more challenging field trial. In the last field test, ClO 2 was generated and routed directly to the scrubber in a 12-h continuous run. Measurement of ClO 2 levels at the generator outlet showed that the desired production rate was not achieved. Additionally, only one of the two scrubbers performed adequately with regard to maintaining ClO 2 emissions below the limit. Numerous lessons were learned in the field trials of this ClO 2 decontamination technology.

  18. Composite sampling of a Bacillus anthracis surrogate with cellulose sponge surface samplers from a nonporous surface.

    Directory of Open Access Journals (Sweden)

    Jenia A M Tufts

    Full Text Available A series of experiments was conducted to explore the utility of composite-based collection of surface samples for the detection of a Bacillus anthracis surrogate using cellulose sponge samplers on a nonporous stainless steel surface. Two composite-based collection approaches were evaluated over a surface area of 3716 cm2 (four separate 929 cm2 areas, larger than the 645 cm2 prescribed by the standard Centers for Disease Control (CDC and Prevention cellulose sponge sampling protocol for use on nonporous surfaces. The CDC method was also compared to a modified protocol where only one surface of the sponge sampler was used for each of the four areas composited. Differences in collection efficiency compared to positive controls and the potential for contaminant transfer for each protocol were assessed. The impact of the loss of wetting buffer from the sponge sampler onto additional surface areas sampled was evaluated. Statistical tests of the results using ANOVA indicate that the collection of composite samples using the modified sampling protocol is comparable to the collection of composite samples using the standard CDC protocol (p  =  0.261. Most of the surface-bound spores are collected on the first sampling pass, suggesting that multiple passes with the sponge sampler over the same surface may be unnecessary. The effect of moisture loss from the sponge sampler on collection efficiency was not significant (p  =  0.720 for both methods. Contaminant transfer occurs with both sampling protocols, but the magnitude of transfer is significantly greater when using the standard protocol than when the modified protocol is used (p<0.001. The results of this study suggest that composite surface sampling, by either method presented here, could successfully be used to increase the surface area sampled per sponge sampler, resulting in reduced sampling times in the field and decreased laboratory processing cost and turn-around times.

  19. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways

    Energy Technology Data Exchange (ETDEWEB)

    Kabilan, S.; Suffield, S. R.; Recknagle, K. P.; Jacob, R. E.; Einstein, D. R.; Kuprat, A. P.; Carson, J. P.; Colby, S. M.; Saunders, J. H.; Hines, S. A.; Teeguarden, J. G.; Straub, T. M.; Moe, M.; Taft, S. C.; Corley, R. A.

    2016-09-01

    Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived respectively from computed tomography (CT) and µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation–exhalation breathing conditions using average species-specific minute volumes. Two different exposure scenarios were modeled in the rabbit based upon experimental inhalation studies. For comparison, human simulations were conducted at the highest exposure concentration used during the rabbit experimental exposures. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the nasal sinus compared to the human at the same air concentration of anthrax spores. In contrast, higher spore deposition was predicted in the lower conducting airways of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology for deposition.

  20. Computational Fluid Dynamics Modeling of Bacillus anthracis Spore Deposition in Rabbit and Human Respiratory Airways

    Energy Technology Data Exchange (ETDEWEB)

    Kabilan, Senthil; Suffield, Sarah R.; Recknagle, Kurtis P.; Jacob, Rick E.; Einstein, Daniel R.; Kuprat, Andrew P.; Carson, James P.; Colby, Sean M.; Saunders, James H.; Hines, Stephanie; Teeguarden, Justin G.; Straub, Tim M.; Moe, M.; Taft, Sarah; Corley, Richard A.

    2016-09-30

    Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived from computed tomography (CT) or µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation-exhalation breathing conditions using average species-specific minute volumes. The highest exposure concentration was modeled in the rabbit based upon prior acute inhalation studies. For comparison, human simulation was also conducted at the same concentration. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the upper conducting airways compared to the human at the same air concentration of anthrax spores. As a result, higher particle deposition was predicted in the conducting airways and deep lung of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology.

  1. Poly(3-hydroxybutyrate) fuels the tricarboxylic acid cycle and de novo lipid biosynthesis during Bacillus anthracis sporulation.

    Science.gov (United States)

    Sadykov, Marat R; Ahn, Jong-Sam; Widhelm, Todd J; Eckrich, Valerie M; Endres, Jennifer L; Driks, Adam; Rutkowski, Gregory E; Wingerd, Kevin L; Bayles, Kenneth W

    2017-06-01

    Numerous bacteria accumulate poly(3-hydroxybutyrate) (PHB) as an intracellular reservoir of carbon and energy in response to imbalanced nutritional conditions. In Bacillus spp., where PHB biosynthesis precedes the formation of the dormant cell type called the spore (sporulation), the direct link between PHB accumulation and efficiency of sporulation was observed in multiple studies. Although the idea of PHB as an intracellular carbon and energy source fueling sporulation was proposed several decades ago, the mechanisms underlying PHB contribution to sporulation have not been defined. Here, we demonstrate that PHB deficiency impairs Bacillus anthracis sporulation through diminishing the energy status of the cells and by reducing carbon flux into the tricarboxylic acid (TCA) cycle and de novo lipid biosynthesis. Consequently, this metabolic imbalance decreased biosynthesis of the critical components required for spore integrity and resistance, such as dipicolinic acid (DPA) and the spore's inner membrane. Supplementation of the PHB deficient mutant with exogenous fatty acids overcame these sporulation defects, highlighting the importance of the TCA cycle and lipid biosynthesis during sporulation. Combined, the results of this work reveal the molecular mechanisms of PHB contribution to B. anthracis sporulation and provide valuable insight into the metabolic requirements for this developmental process in Bacillus species. © 2017 John Wiley & Sons Ltd.

  2. Identification of B. anthracis N(5)-carboxyaminoimidazole ribonucleotide mutase (PurE) active site binding compounds via fragment library screening.

    Science.gov (United States)

    Lei, Hao; Jones, Christopher; Zhu, Tian; Patel, Kavankumar; Wolf, Nina M; Fung, Leslie W-M; Lee, Hyun; Johnson, Michael E

    2016-02-15

    The de novo purine biosynthesis pathway is an attractive target for antibacterial drug design, and PurE from this pathway has been identified to be crucial for Bacillus anthracis survival in serum. In this study we adopted a fragment-based hit discovery approach, using three screening methods-saturation transfer difference nucleus magnetic resonance (STD-NMR), water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR, and surface plasmon resonance (SPR), against B. anthracis PurE (BaPurE) to identify active site binding fragments by initially testing 352 compounds in a Zenobia fragment library. Competition STD NMR with the BaPurE product effectively eliminated non-active site binding hits from the primary hits, selecting active site binders only. Binding affinities (dissociation constant, KD) of these compounds varied between 234 and 301μM. Based on test results from the Zenobia compounds, we subsequently developed and applied a streamlined fragment screening strategy to screen a much larger library consisting of 3000 computationally pre-selected fragments. Thirteen final fragment hits were confirmed to exhibit binding affinities varying from 14μM to 700μM, which were categorized into five different basic scaffolds. All thirteen fragment hits have ligand efficiencies higher than 0.30. We demonstrated that at least two fragments from two different scaffolds exhibit inhibitory activity against the BaPurE enzyme. Published by Elsevier Ltd.

  3. Bacillus anthracis lethal toxin disrupts TCR signaling in CD1d-restricted NKT cells leading to functional anergy.

    Directory of Open Access Journals (Sweden)

    Sunil K Joshi

    2009-09-01

    Full Text Available Exogenous CD1d-binding glycolipid (alpha-Galactosylceramide, alpha-GC stimulates TCR signaling and activation of type-1 natural killer-like T (NKT cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA-mediated intracellular delivery of lethal factor (LF, a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8 and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

  4. Label-Free Detection of Bacillus anthracis Spore Uptake in Macrophage Cells Using Analytical Optical Force Measurements.

    Science.gov (United States)

    Hebert, Colin G; Hart, Sean; Leski, Tomasz A; Terray, Alex; Lu, Qin

    2017-10-03

    Understanding the interaction between macrophage cells and Bacillus anthracis spores is of significant importance with respect to both anthrax disease progression, spore detection for biodefense, as well as understanding cell clearance in general. While most detection systems rely on specific molecules, such as nucleic acids or proteins and fluorescent labels to identify the target(s) of interest, label-free methods probe changes in intrinsic properties, such as size, refractive index, and morphology, for correlation with a particular biological event. Optical chromatography is a label free technique that uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we show an increase in the optical force experienced by RAW264.7 macrophage cells upon the uptake of both microparticles and B. anthracis Sterne 34F2 spores. In the case of spores, the exposure was detected in as little as 1 h without the use of antibodies or fluorescent labels of any kind. An increase in the optical force was also seen in macrophage cells treated with cytochalasin D, both with and without a subsequent exposure to spores, indicating that a portion of the increase in the optical force arises independent of phagocytosis. These results demonstrate the capability of optical chromatography to detect subtle biological differences in a rapid and sensitive manner and suggest future potential in a range of applications, including the detection of biological threat agents for biodefense and pathogens for the prevention of sepsis and other diseases.

  5. Double silencing of relevant genes suggests the existence of the direct link between DNA replication/repair and central carbon metabolism in human fibroblasts.

    Science.gov (United States)

    Wieczorek, Aneta; Fornalewicz, Karolina; Mocarski, Łukasz; Łyżeń, Robert; Węgrzyn, Grzegorz

    2018-04-15

    Genetic evidence for a link between DNA replication and glycolysis has been demonstrated a decade ago in Bacillus subtilis, where temperature-sensitive mutations in genes coding for replication proteins could be suppressed by mutations in genes of glycolytic enzymes. Then, a strong influence of dysfunctions of particular enzymes from the central carbon metabolism (CCM) on DNA replication and repair in Escherichia coli was reported. Therefore, we asked if such a link occurs only in bacteria or it is a more general phenomenon. Here, we demonstrate that effects of silencing (provoked by siRNA) of expression of genes coding for proteins involved in DNA replication and repair (primase, DNA polymerase ι, ligase IV, and topoisomerase IIIβ) on these processes (less efficient entry into the S phase of the cell cycle and decreased level of DNA synthesis) could be suppressed by silencing of specific genes of enzymes from CMM. Silencing of other pairs of replication/repair and CMM genes resulted in enhancement of the negative effects of lower expression levels of replication/repair genes. We suggest that these results may be proposed as a genetic evidence for the link between DNA replication/repair and CMM in human cells, indicating that it is a common biological phenomenon, occurring from bacteria to humans. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. The Saccharomyces cerevisiae MUM2 gene interacts with the DNA replication machinery and is required for meiotic levels of double strand breaks.

    Science.gov (United States)

    Davis, L; Barbera, M; McDonnell, A; McIntyre, K; Sternglanz, R; Jin , Q; Loidl, J; Engebrecht, J

    2001-01-01

    The Saccharomyces cerevisiae MUM2 gene is essential for meiotic, but not mitotic, DNA replication and thus sporulation. Genetic interactions between MUM2 and a component of the origin recognition complex and polymerase alpha-primase suggest that MUM2 influences the function of the DNA replication machinery. Early meiotic gene expression is induced to a much greater extent in mum2 cells than in meiotic cells treated with the DNA synthesis inhibitor hydroxyurea. This result indicates that the mum2 meiotic arrest is downstream of the arrest induced by hydroxyurea and suggests that DNA synthesis is initiated in the mutant. Genetic analyses indicate that the recombination that occurs in mum2 mutants is dependent on the normal recombination machinery and on synaptonemal complex components and therefore is not a consequence of lesions created by incompletely replicated DNA. Both meiotic ectopic and allelic recombination are similarly reduced in the mum2 mutant, and the levels are consistent with the levels of meiosis-specific DSBs that are generated. Cytological analyses of mum2 mutants show that chromosome pairing and synapsis occur, although at reduced levels compared to wild type. Given the near-wild-type levels of meiotic gene expression, pairing, and synapsis, we suggest that the reduction in DNA replication is directly responsible for the reduced level of DSBs and meiotic recombination. PMID:11238403

  7. Failure of Sterne- and Pasteur-like strains of Bacillus anthracis to replicate and survive in the urban bluebottle blow fly Calliphora vicina under laboratory conditions.

    Directory of Open Access Journals (Sweden)

    Britta von Terzi

    Full Text Available This study aimed to elucidate the bacteriological events occurring within the gut of Calliphora vicina, selected as the European representative of blow flies held responsible for the spread of anthrax during epidemics in certain parts of the world. Green-fluorescent-protein-carrying derivatives of Bacillus anthracis were used. These lacked either one of the virulence plasmids pXO1 and pXO2 and were infected, or not infected, with a worm intestine phage (Wip4 known to influence the phenotype and survival of the pathogen. Blood meals were prepared for the flies by inoculation of sheep blood with germinated and, in case of pXO2+ strains, encapsulated cells of the four B. anthracis strains. After being fed for 4 h an initial 10 flies were externally disinfected with peracetic acid to ensure subsequent quantitation representing ingested B. anthracis only. Following neutralization, they were crushed in sterile saline. Over each of the ensuing 7 to 10 days, 10 flies were removed and processed the same way. In the absence of Wip4, strains showed steady declines to undetectable in the total B. anthracis counts, within 7-9 days. With the phage infected strains, the falls in viable counts were significantly more rapid than in their uninfected counterparts. Spores were detectable in flies for longer periods than vegetative bacteria. In line with the findings in both biting and non-biting flies of early workers our results indicate that B. anthracis does not multiply in the guts of blow flies and survival is limited to a matter of days.

  8. Microevolution of Anthrax from a Young Ancestor (M.A.Y.A. Suggests a Soil-Borne Life Cycle of Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Peter Braun

    Full Text Available During an anthrax outbreak at the Pollino National Park (Basilicata, Italy in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA and next generation Whole Genome Sequencing (WGS. PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis.

  9. Naturally acquired antibodies to Bacillus anthracis protective antigen in vultures of southern Africa

    Directory of Open Access Journals (Sweden)

    P. C.B. Turnbull

    2008-08-01

    Full Text Available TURNBULLP, P.C.B. DIEKMANNM,M., KILIAN, J.W., VERSFELDW, W.,DE VOS, V., ARNTZENL, L.,WOLTER, K., BARTELS, P. & KOTZE, A. 2008.N aturally acquired antibodies to Bacillusa nthracisp rotective antigeni n vultureso f southern Africa. Onderstepoort Journal of Veterinary Research, T5:95-102 Sera from 19 wild caught vultures in northern Namibia and 15 (12 wild caught and three captive bred but with minimal histories in North West Province, South Africa, were examined by an enzyme-linked immunosorbenats say( ELISAf or antibodiesto the Bacillus anthracis toxin protective antigen (PA. As assessed from the baseline established with a control group of ten captive reared vultures with well-documented histories, elevated titres were found in 12 of the 19 (63% wild caught Namibian birds as compared with none of the 15 South African ones. There was a highly significant difference between the Namibian group as a hole and the other groups (P 0.05. Numbers in the Namibian group were too small to determine any significances in species-, sex- or age-related differences within the raw data showing elevated titres in four out of six Cape Vultures, Gyps coprotheress, six out of ten Whitebacked Vultures, Gyps africanus, and one out of three Lappet-faced Vultures, Aegypiust racheliotus, or in five of six males versus three of seven females, and ten of 15 adults versus one of four juveniles. The results are in line with the available data on the incidence of anthrax in northern Namibia and South Africa and the likely contact of the vultures tested with anthrax carcasses. lt is not known whether elevated titre indicates infection per se in vultures or absorption of incompletely digested epitopes of the toxin or both. The results are discussed in relation to distances travelled by vultures as determined by new tracking techniques, how serology can reveal anthrax activity in an area and the issue of the role of vultures in transmission of anthrax.

  10. CD4+ T cells targeting dominant and cryptic epitopes from Bacillus anthracis Lethal Factor

    Directory of Open Access Journals (Sweden)

    Stephanie eAscough

    2016-01-01

    Full Text Available Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called ‘cryptic’ or ‘subdominant’ epitopes. We analysed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISPOT assays we characterised epitopes that elicited a response following immunisation with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 trangenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were

  11. Identification of Novel Raft Marker Protein, FlotP in Bacillus anthracis.

    Science.gov (United States)

    Somani, Vikas K; Aggarwal, Somya; Singh, Damini; Prasad, Tulika; Bhatnagar, Rakesh

    2016-01-01

    Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP) harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB) also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homolog harboring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, four were found to harbor RMP. Among them, domains of Bas0525 (FlotP) of Bacillus anthracis (BA) showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA), a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown raft associated

  12. Comparative genomics of Bacillus anthracis from the wool industry highlights polymorphisms of lineage A.Br.Vollum.

    Science.gov (United States)

    Derzelle, Sylviane; Aguilar-Bultet, Lisandra; Frey, Joachim

    2016-12-01

    With the advent of affordable next-generation sequencing (NGS) technologies, major progress has been made in the understanding of the population structure and evolution of the B. anthracis species. Here we report the use of whole genome sequencing and computer-based comparative analyses to characterize six strains belonging to the A.Br.Vollum lineage. These strains were isolated in Switzerland, in 1981, during iterative cases of anthrax involving workers in a textile plant processing cashmere wool from the Indian subcontinent. We took advantage of the hundreds of currently available B. anthracis genomes in public databases, to investigate the genetic diversity existing within the A.Br.Vollum lineage and to position the six Swiss isolates into the worldwide B. anthracis phylogeny. Thirty additional genomes related to the A.Br.Vollum group were identified by whole-genome single nucleotide polymorphism (SNP) analysis, including two strains forming a new evolutionary branch at the basis of the A.Br.Vollum lineage. This new phylogenetic lineage (termed A.Br.H9401) splits off the branch leading to the A.Br.Vollum group soon after its divergence to the other lineages of the major A clade (i.e. 6 SNPs). The available dataset of A.Br.Vollum genomes were resolved into 2 distinct groups. Isolates from the Swiss wool processing facility clustered together with two strains from Pakistan and one strain of unknown origin isolated from yarn. They were clearly differentiated (69 SNPs) from the twenty-five other A.Br.Vollum strains located on the branch leading to the terminal reference strain A0488 of the lineage. Novel analytic assays specific to these new subgroups were developed for the purpose of rapid molecular epidemiology. Whole genome SNP surveys greatly expand upon our knowledge on the sub-structure of the A.Br.Vollum lineage. Possible origin and route of spread of this lineage worldwide are discussed. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights

  13. Residual DNA analysis in biologics development: review of measurement and quantitation technologies and future directions.

    Science.gov (United States)

    Wang, Xing; Morgan, Donna M; Wang, Gan; Mozier, Ned M

    2012-02-01

    Residual DNA (rDNA) is comprised of deoxyribonucleic acid (DNA) fragments and longer length molecules originating from the host organism that may be present in samples from recombinant biological processes. Although similar in basic structural base pair units, rDNA may exist in different sizes and physical forms. Interest in measuring rDNA in recombinant products is based primarily on demonstration of effective purification during manufacturing, but also on some hypothetical concerns that, in rare cases, depending on the host expression system, some DNA sequences may be potentially infectious or oncogenic (e.g., HIV virus and the Ras oncogene, respectively). Recent studies suggest that a sequence known as long interspersed nucleotide element-1 (LINE-1), widely distributed in the mammalian genome, is active as a retrotransposon that can be transcribed to RNA, reverse-transcribed into DNA and inserts into a new site in genome. This integration process could potentially disrupt critical gene functions or induce tumorigenesis in mammals. Genomic DNA from microbial sources, on the other hand, could add to risk of immunogenicity to the target recombinant protein being expressed, due to the high CpG content and unmethylated DNA sequence. For these and other reasons, it is necessary for manufacturers to show clearance of DNA throughout production processes and to confirm low levels in the final drug substance using an appropriately specific and quantitative analytical method. The heterogeneity of potential rDNA sequences that might be makes the testing of all potential analytes challenging. The most common methodology for rDNA quantitation used currently is real-time polymerase chain reaction (RT-PCR), a robust and proven technology. Like most rDNA quantitation methods, the specificity of RT-PCR is limited by the sequences to which the primers are directed. To address this, primase-based whole genome amplification is introduced herein. This paper will review the recent

  14. A strain-variable bacteriocin in Bacillus anthracis and Bacillus cereus with repeated Cys-Xaa-Xaa motifs

    Directory of Open Access Journals (Sweden)

    Haft Daniel H

    2009-04-01

    Full Text Available Abstract Bacteriocins are peptide antibiotics from ribosomally translated precursors, produced by bacteria often through extensive post-translational modification. Minimal sequence conservation, short gene lengths, and low complexity sequence can hinder bacteriocin identification, even during gene calling, so they are often discovered by proximity to accessory genes encoding maturation, immunity, and export functions. This work reports a new subfamily of putative thiazole-containing heterocyclic bacteriocins. It appears universal in all strains of Bacillus anthracis and B. cereus, but has gone unrecognized because it is always encoded far from its maturation protein operon. Patterns of insertions and deletions among twenty-four variants suggest a repeating functional unit of Cys-Xaa-Xaa. Reviewers This article was reviewed by Andrei Osterman and Lakshminarayan Iyer.

  15. The central nervous system as target of Bacillus anthracis toxin independent virulence in rabbits and guinea pigs.

    Directory of Open Access Journals (Sweden)

    Haim Levy

    Full Text Available Infection of the central nervous system is considered a complication of Anthrax and was reported in humans and non-human primates. Previously we have reported that Bacillus anthracis possesses a toxin-independent virulent trait that, like the toxins, is regulated by the major virulence regulator, AtxA, in the presence of pXO2. This toxin-independent lethal trait is exhibited in rabbits and Guinea pigs following significant bacteremia and organ dissemination. Various findings, including meningitis seen in humans and primates, suggested that the CNS is a possible target for this AtxA-mediated activity. In order to penetrate into the brain tissue, the bacteria have to overcome the barriers isolating the CNS from the blood stream. Taking a systematic genetic approach, we compared intracranial (IC inoculation and IV/SC inoculation for the outcome of the infection in rabbits/GP, respectively. The outstanding difference between the two models is exhibited by the encapsulated strain VollumΔpXO1, which is lethal when injected IC, but asymptomatic when inoculated IV/SC. The findings demonstrate that there is an apparent bottleneck in the ability of mutants to penetrate into the brain. Any mutant carrying either pXO1 or pXO2 will kill the host upon IC injection, but only those carrying AtxA either on pXO1 or in the chromosome in the background of pXO2 can penetrate into the brain following peripheral inoculation. The findings were corroborated by histological examination by H&E staining and immunofluorescence of rabbits' brains following IV and IC inoculations. These findings may have major implications on future research both on B. anthracis pathogenicity and on vaccine development.

  16. CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

    Science.gov (United States)

    Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

    2014-10-28

    DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

  17. Cloning and Expression of Fusion Genes of Domain A-1 Protective Antigen of Bacillus Anthracis and Shigella Enterotoxin B Subunit (Stxb In E. Coil

    Directory of Open Access Journals (Sweden)

    AH ahmadi

    2015-02-01

    Conclusion: The findings of the current study revealed that this antigen can be raised as an anti-cancer and recombinant vaccine candidate against types of Shigella, Escherichia coli and Bacillus anthracis which can be due to such factors as identification of antigen(PA by antibody PA20, its apoptosis induction properties, property of immunogenicity, adjuvant and delivery of STxB protein and high expression levels of Gb3 in human cancer cells.

  18. Identification of Ciprofloxacin Resistance by SimpleProbe (trademark), High Resolution Melt and Pyrosequencing (trademark) Nucleic Acid Analysis in Biothreat Agents: Bacillus anthracis, Yersinia pestis and Francisella tularensis

    Science.gov (United States)

    2010-01-01

    tularensis strain Schu4 following standardmicrobiological techniques to develop antibiotic resistance. Colonies from an original chocolate agar culture...35 C for two weeks. Growth in broth containing 16 mg/ml ciprofloxacin was transferred to chocolate agar plates containing 64 mg/ml ciprofloxacin and...expeditious therapy , the three PCR technologies described in this study were evaluated for the ability to accurately differentiate wt B. anthracis, Y

  19. Ebselen and analogs as inhibitors of Bacillus anthracis thioredoxin reductase and bactericidal antibacterials targeting Bacillus species, Staphylococcus aureus and Mycobacterium tuberculosis.

    Science.gov (United States)

    Gustafsson, Tomas N; Osman, Harer; Werngren, Jim; Hoffner, Sven; Engman, Lars; Holmgren, Arne

    2016-06-01

    Bacillus anthracis is the causative agent of anthrax, a disease associated with a very high mortality rate in its invasive forms. We studied a number of ebselen analogs as inhibitors of B. anthracis thioredoxin reductase and their antibacterial activity on Bacillus subtilis, Staphylococcus aureus, Bacillus cereus and Mycobacterium tuberculosis. The most potent compounds in the series gave IC(50) values down to 70 nM for the pure enzyme and minimal inhibitory concentrations (MICs) down to 0.4 μM (0.12 μg/ml) for B. subtilis, 1.5 μM (0.64 μg/ml) for S. aureus, 2 μM (0.86 μg/ml) for B. cereus and 10 μg/ml for M. tuberculosis. Minimal bactericidal concentrations (MBCs) were found at 1-1.5 times the MIC, indicating a general, class-dependent, bactericidal mode of action. The combined bacteriological and enzymological data were used to construct a preliminary structure-activity-relationship for the benzoisoselenazol class of compounds. When S. aureus and B. subtilis were exposed to ebselen, we were unable to isolate resistant mutants on both solid and in liquid medium suggesting a high resistance barrier. These results suggest that ebselen and analogs thereof could be developed into a novel antibiotic class, useful for the treatment of infections caused by B. anthracis, S. aureus, M. tuberculosis and other clinically important bacteria. Furthermore, the high barrier against resistance development is encouraging for further drug development. We have characterized the thioredoxin system from B. anthracis as a novel drug target and ebselen and analogs thereof as a potential new class of antibiotics targeting several important human pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvate

    International Nuclear Information System (INIS)

    Voss, Jarrod E.; Scally, Stephen W.; Taylor, Nicole L.; Dogovski, Con; Alderton, Malcolm R.; Hutton, Craig A.; Gerrard, Juliet A.; Parker, Michael W.; Dobson, Renwick C. J.; Perugini, Matthew A.

    2009-01-01

    Dihydrodipicolinate synthase (DHDPS) catalyses an important step in lysine biosynthesis. Here, the expression, purification, crystallization and preliminary diffraction analysis to 2.15 Å resolution of DHDPS from B. anthracis soaked with the substrate pyruvate are reported. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the lysine-biosynthesis pathway in bacteria, plants and some fungi. In this study, the expression of DHDPS from Bacillus anthracis (Ba-DHDPS) and the purification of the recombinant enzyme in the absence and presence of the substrate pyruvate are described. It is shown that DHDPS from B. anthracis purified in the presence of pyruvate yields greater amounts of recombinant enzyme with more than 20-fold greater specific activity compared with the enzyme purified in the absence of substrate. It was therefore sought to crystallize Ba-DHDPS in the presence of the substrate. Pyruvate was soaked into crystals of Ba-DHDPS prepared in 0.2 M sodium fluoride, 20%(w/v) PEG 3350 and 0.1 M bis-tris propane pH 8.0. Preliminary X-ray diffraction data of the recombinant enzyme soaked with pyruvate at a resolution of 2.15 Å are presented. The pending crystal structure of the pyruvate-bound form of Ba-DHDPS will provide insight into the function and stability of this essential bacterial enzyme

  1. Discrimination of Bacillus anthracis Spores by Direct in-situ Analysis of Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

    International Nuclear Information System (INIS)

    Jeong, Youngsu; Lee, Jonghee; Kim, Seongsoo

    2013-01-01

    The rapid and accurate identification of biological agents is a critical step in the case of bio-terror and biological warfare attacks. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been widely used for the identification of microorganisms. In this study, we describe a method for the rapid and accurate discrimination of Bacillus anthracis spores using MALDI-TOF MS. Our direct in-situ analysis of MALDI-TOF MS does not involve subsequent high-resolution mass analyses and sample preparation steps. This method allowed the detection of species-specific biomarkers from each Bacillus spores. Especially, B. anthracis spores had specific biomarker peaks at 2503, 3089, 3376, 6684, 6698, 6753, and 6840 m/z. Cluster and PCA analyses of the mass spectra of Bacillus spores revealed distinctively separated clusters and within-groups similarity. Therefore, we believe that this method is effective in the real-time identification of biological warfare agents such as B. anthracis as well as other microorganisms in the field

  2. A survey of the occurrence of Bacillus anthracis in North American soils over two long-range transects and within post-Katrina New Orleans

    Science.gov (United States)

    Griffin, Dale W.; Petrosky, Terry; Morman, Suzette A.; Luna, Vicki A.

    2009-01-01

    Soil samples were collected along a north-south transect extending from Manitoba, Canada, to the US-Mexico border near El Paso, Texas in 2004 (104 samples), a group of sites within New Orleans, Louisiana following Hurricane Katrina in 2005 (19 samples), and a Gulf Coast transect extending from Sulphur, Louisiana, to DeFuniak Springs, Florida, in 2007 (38 samples). Samples were collected from the top 40 cm of soil and were screened for the presence of total Bacillus species and Bacillus anthracis (anthrax), specifically using multiplex-polymerase chain reaction (PCR). Using an assay with a sensitivity of ~170 equivalent colony-forming units (CFU) g-1 field moist soil, the prevalence rate of Bacillus sp./B. anthracis in the north-south transect and the 2005 New Orleans post-Katrina sample set were 20/5% and 26/26%, respectively. Prevalence in the 2007 Gulf Coast sample set using an assay with a sensitivity of ~4 CFU g-1 of soil was 63/0%. Individual transect-set data indicate a positive relation between occurrences of species and soil moisture or soil constituents (i.e., Zn and Cu content). The 2005 New Orleans post-Katrina data indicated that B. anthracis is readily detectable in Gulf Coast soils following flood events. The data also indicated that occurrence, as it relates to soil chemistry, may be confounded by flood-induced dissemination of germinated cells and the mixing of soil constituents for short temporal periods following an event.

  3. Discrimination of Bacillus anthracis Spores by Direct in-situ Analysis of Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Youngsu; Lee, Jonghee; Kim, Seongsoo [Agency for Defense Development, Daejeon (Korea, Republic of)

    2013-09-15

    The rapid and accurate identification of biological agents is a critical step in the case of bio-terror and biological warfare attacks. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been widely used for the identification of microorganisms. In this study, we describe a method for the rapid and accurate discrimination of Bacillus anthracis spores using MALDI-TOF MS. Our direct in-situ analysis of MALDI-TOF MS does not involve subsequent high-resolution mass analyses and sample preparation steps. This method allowed the detection of species-specific biomarkers from each Bacillus spores. Especially, B. anthracis spores had specific biomarker peaks at 2503, 3089, 3376, 6684, 6698, 6753, and 6840 m/z. Cluster and PCA analyses of the mass spectra of Bacillus spores revealed distinctively separated clusters and within-groups similarity. Therefore, we believe that this method is effective in the real-time identification of biological warfare agents such as B. anthracis as well as other microorganisms in the field.

  4. Structures of an alanine racemase from Bacillus anthracis (BA0252) in the presence and absence of (R)-1-aminoethylphosphonic acid (l-Ala-P)

    International Nuclear Information System (INIS)

    Au, Kinfai; Ren, Jingshan; Walter, Thomas S.; Harlos, Karl; Nettleship, Joanne E.; Owens, Raymond J.; Stuart, David I.; Esnouf, Robert M.

    2008-01-01

    Structures of BA0252, an alanine racemase from B. anthracis, in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (l-Ala-P) and determined by X-ray crystallography to resolutions of 2.1 and 1.47 Å, respectively, are described. Bacillus anthracis, the causative agent of anthrax, has been targeted by the Oxford Protein Production Facility to validate high-throughput protocols within the Structural Proteomics in Europe project. As part of this work, the structures of an alanine racemase (BA0252) in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (l-Ala-P) have determined by X-ray crystallo@@graphy to resolutions of 2.1 and 1.47 Å, respectively. Difficulties in crystallizing this protein were overcome by the use of reductive methylation. Alanine racemase has attracted much interest as a possible target for anti-anthrax drugs: not only is d-alanine a vital component of the bacterial cell wall, but recent studies also indicate that alanine racemase, which is accessible in the exosporium, plays a key role in inhibition of germination in B. anthracis. These structures confirm the binding mode of l-Ala-P but suggest an unexpected mechanism of inhibition of alanine racemase by this compound and could provide a basis for the design of improved alanine racemase inhibitors with potential as anti-anthrax therapies

  5. [Development and comparative evaluation of up-converting phosphor technology based lateral flow assay for rapid detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp].

    Science.gov (United States)

    Li, Chunfeng; Zhang, Pingping; Wang, Xiaoying; Liu, Xiao; Zhao, Yong; Sun, Chongyun; Wang, Chengbin; Yang, Ruifu; Zhou, Lei

    2015-01-01

    To develop an up-converting phosphor technology based lateral flow (UPT-LF) assay for rapid and quantitative detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp.and make the comparison with BioThreat Alert (BTA) test strips (Tetracore Inc., USA). Using up-converting phosphor nano-particles (UCP-NPs) as the bio-marker, three double-antibody-sandwich model based UPT-LF strips including Plague-UPT-LF, Anthrax-UPT-LF, Brucella-UPT-LF were prepared and its sensitivity, accuracy, linearity and specificity were determined by detecting 10(10), 10(9), 10(8), 10(7), 10(6), 10(5) and 0 CFU/ml series of concentrations of Y.pestis, B.anthracis, Brucella standards and other 27 kinds of 10(9) CFU/ml series of contrations of bacteria strains.Furthermore, the speed, sensitivity and accuracy of bacteria standards and simulated sample detection were compared between UPT-LF and BTA system. The detection limit of Plague-UPT-LF, Anthrax-UPT-LF and Brucella-LF was 10(5) CFU/ml. The CV of series of bacteria concentrations was ≤ 15%, and the r between lg (T/C-cut-off) and lg (concentration) was 0.996,0.998 and 0.999 (F values were 1 647.57, 743.51 and 1 822.17. All the P values were Brucella-LF were excellent, while that of Anthrax-UPT-LF was a little bit regretful because of non-specific reaction with two isolates of B. subtilis and one B.cereus. On-site evaluation showed the detection time of UPT-LF for all Y.pestis, B.anthracis spore and Brucella spp.was 33, 36 and 37 min, while BTA was 115, 115 and 111 min, which revealed the higher detection speed and sensitivity of UPT-LF comparing with BTA. The negative rate of two methods for blank standard was both 5/5, the sensitivity of UPT-LF for Y.pestis,B.anthracis spore and Brucella spp. was all 10(5) CFU/ml, then BTA was 10(6), 10(6) and 10(5) CFU/ml, respectively. The detection rate of UPT-LF for all three bacteria analog positive samples was 16/16, while BTA for B.anthracis was 7/16 only. The good performance

  6. A New FRET-Based Sensitive DNA Sensor for Medical Diagnostics using PNA Probe and Water-Soluble Blue Light Emitting Polymer

    Directory of Open Access Journals (Sweden)

    Nidhi Mathur

    2008-01-01

    Full Text Available A reliable, fast, and low-cost biosensor for medical diagnostics using DNA sequence detection has been developed and tested for the detection of the bacterium “Bacillus anthracis.” In this sensor, Poly [9,9-di (6,6′- N, N′ trimethylammonium hexylfluorenyl-2, 7-diyl-alt-co- (1,4-phenylene] dibromide salt (PFP has been taken as cationic conjugated polymer (CCP and PNA attached with fluorescein dye (PNAC∗ as a probe. The basic principle of this sensor is that when a PNAC∗ probe is hybridized with a single strand DNA (ssDNA having complementary sequence, Forster resonance energy transfer (FRET may take place from PFP to the PNAC∗/DNA complex. If the FRET is efficient, the photoluminescence from the PFP will be highly quenched and that from PNAC∗ will be enhanced. On the other hand, if the DNA sequence is noncomplementary to PNA, FRET will not occur.

  7. Bacillus anthracis o-succinylbenzoyl-CoA synthetase: reaction kinetics and a novel inhibitor mimicking its reaction intermediate.

    Science.gov (United States)

    Tian, Yang; Suk, Dae-Hwan; Cai, Feng; Crich, David; Mesecar, Andrew D

    2008-11-25

    o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered bi uni uni bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first half-reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the "F" form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered bi uni uni bi iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogues with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogues tested as none were active except 4-[2-(trifluoromethyl)phenyl]-4-oxobutyric acid which exhibited a 100-fold decrease in k(cat)/K(m). On the basis of an understanding of OSB-CoA synthetase's kinetic mechanism and substrate specificity, a reaction intermediate analogue of OSB-AMP, 5'-O-{N-[2-(trifluoromethyl)phenyl]-4-oxobutyl}adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase

  8. Bacillus anthracis o-succinylbenzoyl-CoA synthetase: reaction kinetics and a novel inhibitor mimicking its reaction intermediate †

    Science.gov (United States)

    Tian, Yang; Suk, Dae-Hwan; Cai, Feng; Crich, David; Mesecar, Andrew D.

    2009-01-01

    O-succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered Bi Uni Uni Bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct-binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first-half reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the ‘F’ form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered Bi Uni Uni Bi Iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogs with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogs tested as none were active except 4-(2-trifluoromethylphenyl)-4-oxobutyric acid which exhibited a 100-fold decrease in kcat/Km. Based on an understanding of OSB-CoA synthetase’s kinetic mechanism and substrate specificity, a reaction intermediate analog of OSB-AMP, 5’-O-(N-(2-trifluoromethylphenyl)-4-oxobutyl) adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase class of

  9. Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture

    Energy Technology Data Exchange (ETDEWEB)

    Hutchison, J. R. [National Security Directorate, Pacific Northwest National Laboratory, Richland WA USA; Piepel, G. F. [National Security Directorate, Pacific Northwest National Laboratory, Richland WA USA; Amidan, B. G. [National Security Directorate, Pacific Northwest National Laboratory, Richland WA USA; Hess, B. M. [National Security Directorate, Pacific Northwest National Laboratory, Richland WA USA; Sydor, M. A. [National Security Directorate, Pacific Northwest National Laboratory, Richland WA USA; Deatherage Kaiser, B. L. [National Security Directorate, Pacific Northwest National Laboratory, Richland WA USA

    2018-03-13

    Aims: We evaluated the effects of Bacillus anthracis surrogates, low surface concentrations, surface materials, and assay methods on false-negative rate (FNR) and limit of detection (LOD95) for recovering Bacillus spores using a macrofoam-swab sampling procedure. Methods and Results: Bacillus anthracis Sterne or Bacillus atrophaeus Nakamura spores were deposited over a range of low target concentrations (2 – 500 coupon-1) onto glass, stainless steel, vinyl tile, and plastic. Samples were assayed using a modified Rapid Viability-PCR (mRV-PCR) method and the traditional plate culture method to obtain FNR and LOD95 results. Conclusions: Mean FNRs tended to be lower for mRV-PCR compared to culturing, and increased as spore concentration decreased for all surface materials. Surface material, but not B. anthracis surrogate, influenced FNRs with the mRV-PCR method. The mRV-PCR LOD95 was lowest for glass and highest for vinyl tile. LOD95 values overall were lower for mRV-PCR than for the culture method. Significance and Impact of Study: This study adds to the limited data on FNR and LOD95 for mRV-PCR and culturing methods with low concentrations of B. anthracis sampled from various surface materials by the CDC macrofoam-swab method. These are key inputs for planning characterization and clearance studies for low contamination levels of B. anthracis.

  10. CD and MCD spectroscopic studies of the two Dps miniferritin proteins from Bacillus anthracis: role of O2 and H2O2 substrates in reactivity of the diiron catalytic centers.

    Science.gov (United States)

    Schwartz, Jennifer K; Liu, Xiaofeng S; Tosha, Takehiko; Diebold, Adrienne; Theil, Elizabeth C; Solomon, Edward I

    2010-12-14

    DNA protection during starvation (Dps) proteins are miniferritins found in bacteria and archaea that provide protection from uncontrolled Fe(II)/O radical chemistry; thus the catalytic sites are targets for antibiotics against pathogens, such as anthrax. Ferritin protein cages synthesize ferric oxymineral from Fe(II) and O(2)/H(2)O(2), which accumulates in the large central cavity; for Dps, H(2)O(2) is the more common Fe(II) oxidant contrasting with eukaryotic maxiferritins that often prefer dioxygen. To better understand the differences in the catalytic sites of maxi- versus miniferritins, we used a combination of NIR circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) to study Fe(II) binding to the catalytic sites of the two Bacillus anthracis miniferritins: one in which two Fe(II) react with O(2) exclusively (Dps1) and a second in which both O(2) or H(2)O(2) can react with two Fe(II) (Dps2). Both result in the formation of iron oxybiomineral. The data show a single 5- or 6-coordinate Fe(II) in the absence of oxidant; Fe(II) binding to Dps2 is 30× more stable than Dps1; and the lower limit of K(D) for binding a second Fe(II), in the absence of oxidant, is 2-3 orders of magnitude weaker than for the binding of the single Fe(II). The data fit an equilibrium model where binding of oxidant facilitates formation of the catalytic site, in sharp contrast to eukaryotic M-ferritins where the binuclear Fe(II) centers are preformed before binding of O(2). The two different binding sequences illustrate the mechanistic range possible for catalytic sites of the family of ferritins.

  11. Differential Binding of Co(II) and Zn(II) to Metallo-beta-Lactamase Bla2 from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Hawk, M.; Breece, R; Hajdin, C; Bender, K; Hu, Z; Costello, A; Bennett, B; Tierney, D; Crowder, M

    2009-01-01

    In an effort to probe the structure, mechanism, and biochemical properties of metallo-{beta}-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV-vis, 1H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms.

  12. Obiltoxaximab Prevents Disseminated Bacillus anthracis Infection and Improves Survival during Pre- and Postexposure Prophylaxis in Animal Models of Inhalational Anthrax

    Science.gov (United States)

    Yamamoto, Brent J.; Shadiack, Annette M.; Carpenter, Sarah; Sanford, Daniel; Henning, Lisa N.; Gonzales, Nestor; O'Connor, Edward; Casey, Leslie S.

    2016-01-01

    The Centers for Disease Control and Prevention recommend adjunctive antitoxins when systemic anthrax is suspected. Obiltoxaximab, a monoclonal antibody against protective antigen (PA), is approved for treatment of inhalational anthrax in combination with antibiotics and for prophylaxis when alternative therapies are not available. The impact of toxin neutralization with obiltoxaximab during pre- and postexposure prophylaxis was explored, and efficacy results that supported the prophylaxis indication are presented here. New Zealand White rabbits and cynomolgus macaques received obiltoxaximab as a single intramuscular or intravenous dose of 2 to 16 mg/kg of body weight at various times relative to Bacillus anthracis aerosol spore challenge. The primary endpoint was survival, and effect of treatment timing was explored. In rabbits, obiltoxaximab administration 9 h postchallenge singly or combined with a 5-day levofloxacin regimen protected 89% to 100% of animals compared to 33% with levofloxacin monotherapy. In cynomolgus macaques, a single intramuscular dose of 16 mg/kg obiltoxaximab led to 100% survival when given 1 to 3 days preexposure and 83% to 100% survival when given 18 to 24 h postexposure and prior to systemic bacteremia onset. Obiltoxaximab administration after bacteremia onset resulted in lower (25% to 50%) survival rates reflective of treatment setting. Prophylactic administration of obiltoxaximab before spore challenge or to spore-challenged animals before systemic bacterial dissemination is efficacious in promoting survival, ameliorating toxemia, and inhibiting bacterial spread to the periphery. PMID:27431219

  13. BLACK-BACKED JACKAL EXPOSURE TO RABIES VIRUS, CANINE DISTEMPER VIRUS, AND BACILLUS ANTHRACIS IN ETOSHA NATIONAL PARK, NAMIBIA

    Science.gov (United States)

    Bellan, Steve E.; Cizauskas, Carrie A.; Miyen, Jacobeth; Ebersohn, Karen; Küsters, Martina; Prager, Katie; Van Vuuren, Moritz; Sabeta, Claude; Getz, Wayne M.

    2017-01-01

    Canine distemper virus (CDV) and rabies virus (RABV) occur worldwide in wild carnivore and domestic dog populations and pose threats to wildlife conservation and public health. In Etosha National Park (ENP), Namibia, anthrax is endemic and generates carcasses frequently fed on by an unusually dense population of black-backed jackals (Canis mesomelas). Using serology and phylogenetic analyses (on samples obtained from February, 2009 to July, 2010), and historical mortality records (1975–2011), we assessed jackal exposure to Bacillus anthracis (BA; the causal bacterial agent of anthrax), CDV, and RABV. Seroprevalence to all three pathogens was relatively high with 95% (n = 86), 73% (n = 86), and 9% (n = 81) of jackals exhibiting antibodies to BA, CDV, and RABV, respectively. Exposure to BA, as assessed with an anti-Protective Antigen ELISA test, increased significantly with age and all animals >1 yr old tested positive. Seroprevalence of exposure to CDV also increased significantly with age, with similar age-specific trends during both years of the study. No significant effect of age was found on RABV seroprevalence. Three of the seven animals exhibiting immunity to RABV were monitored for more than one year after sampling and did not succumb to the disease. Mortality records revealed that rabid animals are destroyed nearly every year inside the ENP tourist camps. Phylogenetic analyses demonstrated that jackal RABV in ENP is part of the same transmission cycle as other dog-jackal RABV cycles in Namibia. PMID:22493112

  14. Biochemical and Structural Analysis of an Eis Family Aminoglycoside Acetyltransferase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Green, Keith D.; Biswas, Tapan; Chang, Changsoo; Wu, Ruiying; Chen, Wenjing; Janes, Brian K.; Chalupska, Dominika; Gornicki, Piotr; Hanna, Philip C.; Tsodikov, Oleg V.; Joachimiak, Andrzej; Garneau-Tsodikova, Sylvie

    2015-05-26

    Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis_Mtb) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis_Ban). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis_Ban displays significant differences from Eis_Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was also different for several AGs, indicating a distinct regiospecificity of Eis_Ban. Furthermore, most recently identified inhibitors of Eis_Mtb did not inhibit Eis_Ban, underscoring the differences between these two enzymes. To explain these differences, we determined an Eis_Ban crystal structure. The comparison of the crystal structures of Eis_Ban and Eis_Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance.

  15. Modeling DNA

    Science.gov (United States)

    Robertson, Carol

    2016-01-01

    Deoxyribonucleic acid (DNA) is life's most amazing molecule. It carries the genetic instructions that almost every organism needs to develop and reproduce. In the human genome alone, there are some three billion DNA base pairs. The most difficult part of teaching DNA structure, however, may be getting students to visualize something as small as a…

  16. False Negative Rates of a Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates via Real-Time PCR

    Energy Technology Data Exchange (ETDEWEB)

    Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Amidan, Brett G. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sydor, Michael A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Kaiser, Brooke L.D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-06-01

    Surface sampling for Bacillus anthracis spores has traditionally relied on detection via bacterial cultivation methods. Although effective, this approach does not provide the level of organism specificity that can be gained through molecular techniques. False negative rates (FNR) and limits of detection (LOD) were determined for two B. anthracis surrogates with modified rapid viability-polymerase chain reaction (mRV-PCR) following macrofoam-swab sampling. This study was conducted in parallel with a previously reported study that analyzed spores using a plate-culture method. B. anthracis Sterne (BAS) or B. atrophaeus Nakamura (BG) spores were deposited onto four surface materials (glass, stainless steel, vinyl tile, and plastic) at nine target concentrations (2 to 500 spores/coupon; 0.078 to 19.375 colony-forming units [CFU] per cm2). Mean FNR values for mRV-PCR analysis ranged from 0 to 0.917 for BAS and 0 to 0.875 for BG and increased as spore concentration decreased (over the concentrations investigated) for each surface material. FNRs based on mRV-PCR data were not statistically different for BAS and BG, but were significantly lower for glass than for vinyl tile. FNRs also tended to be lower for the mRV-PCR method compared to the culture method. The mRV-PCR LOD95 was lowest for glass (0.429 CFU/cm2 with BAS and 0.341 CFU/cm2 with BG) and highest for vinyl tile (0.919 CFU/cm2 with BAS and 0.917 CFU/cm2 with BG). These mRV-PCR LOD95 values were lower than the culture values (BAS: 0.678 to 1.023 CFU/cm2 and BG: 0.820 to 1.489 CFU/cm2). The FNR and LOD95 values reported in this work provide guidance for environmental sampling of Bacillus spores at low concentrations.

  17. Comparison of French and Worldwide Bacillus anthracis Strains Favors a Recent, Post-Columbian Origin of the Predominant North-American Clade.

    Science.gov (United States)

    Vergnaud, Gilles; Girault, Guillaume; Thierry, Simon; Pourcel, Christine; Madani, Nora; Blouin, Yann

    2016-01-01

    Bacillus anthracis, the highly dangerous zoonotic bacterial pathogen species is currently composed of three genetic groups, called A, B and C. Group A is represented worldwide whereas group B is present essentially in Western Europe and Southern Africa. Only three strains from group C have been reported. This knowledge is derived from the genotyping of more than 2000 strains collected worldwide. Strains from both group A and group B are present in France. Previous investigations showed that the majority of sporadic French strains belong to the so-called A.Br.011/009 group A clade and define a very remarkable polytomy with six branches. Here we explore the significance of this polytomy by comparing the French B. anthracis lineages to worldwide lineages. We take advantage of whole genome sequence data previously determined for 122 French strains and 45 strains of various origins. A total of 6690 SNPs was identified among the available dataset and used to draw the phylogeny. The phylogeny of the French B group strains which belongs to B.Br.CNEVA indicates an expansion from the south-east part of France (the Alps) towards the south-west (Massif-Central and Pyrenees). The relatively small group A strains belonging to A.Br.001/002 results from at least two independent introductions. Strikingly, the data clearly demonstrates that the currently predominant B. anthracis lineage in North America, called WNA for Western North American, is derived from one branch of the A.Br.011/009 polytomy predominant in France. The present work extends the range of observed substitution rate heterogeneity within B. anthracis, in agreement with its ecology and in contrast with some other pathogens. The population structure of the six branches A.Br.011/009 polytomy identified in France, diversity of branch length, and comparison with the WNA lineage, suggests that WNA is of post-Columbian and west European origin, with France as a likely source. Furthermore, it is tempting to speculate that

  18. Bacterial spores as possible contaminants of biomedical materials and devices. [Bacillus anthracis, clostridium botulinum, C. perfringens, C. tetani

    Energy Technology Data Exchange (ETDEWEB)

    Grecz, N; Kang, T

    1973-01-01

    Destruction of spores on biomedical devices in drugs, and biologicals is essential for prevention of infection of patients with pathogenic sporeformers. Of particular concern are Clostridium tetani, C. perfringens, C. botulinum, Bacillus anthracis and other sporeforming pathogens. Spores are ubiquitous in nature and contamination of biomedical devices varies depending on manufacturing process, handling, raw materials and other variables. In the last 20 years the number of cases per year of specific notifiable diseases in the United States was as follows: tetanus, 120 to 500 cases, botulism, 7 to 47 cases, and anthrax, 2 to 10 cases. Gas gangrene is caused by a mixed flora consisting predominantly of sporeformers. C botulinum, which usually acts as saprophytic agent of food poisoning, may also initiate pathogenic processes; there are nine cases on record in the United States of botulism wound infections almost half of which ended in death. The spores of these organisms are distinguished by high radiation resistance and their erradication often requires severe radiation treatments. Representative bacterial spores in various suspending media show D/sub 10/ values (dose necessary to destroy 90 percent of a given population) ranging from approximately 0.1 to 0.4 Mrad. Some viruses show D/sub 10/ values up to greater than 1 Mrad. The D/sub 10/-values of spores vary depending on physical, chemical and biological factors. This variability is important in evaluation and selection of biological indicator organisms. Radiation sterilization of biomedical devices and biomedical materials must provide safety from infectious microorganisms including radiation resistant spores and viruses.

  19. Passive vaccination with a human monoclonal antibody: generation of antibodies and studies for efficacy in Bacillus anthracis infections.

    Science.gov (United States)

    vor dem Esche, Ulrich; Huber, Maria; Zgaga-Griesz, Andrea; Grunow, Roland; Beyer, Wolfgang; Hahn, Ulrike; Bessler, Wolfgang G

    2011-07-01

    A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121-150 or 451-470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination. Copyright © 2010 Elsevier GmbH. All rights reserved.

  20. DNA Camouflage

    Science.gov (United States)

    2016-01-08

    1 DNA Camouflage Supplementary Information Bijan Zakeri1,2*, Timothy K. Lu1,2*, Peter A. Carr2,3* 1Department of Electrical Engineering and...ll.mit.edu). Distribution A: Public Release   2 Supplementary Figure 1 DNA camouflage with the 2-state device. (a) In the presence of Cre, DSD-2[α...10 1 + Cre 1 500 1,000 length (bp) chromatogram alignment template − Cre   4 Supplementary Figure 3 DNA camouflage with a switchable

  1. Decontamination of materials contaminated with Bacillus anthracis and Bacillus thuringiensis Al Hakam spores using PES-Solid, a solid source of peracetic acid.

    Science.gov (United States)

    Buhr, T L; Wells, C M; Young, A A; Minter, Z A; Johnson, C A; Payne, A N; McPherson, D C

    2013-08-01

    To develop test methods and evaluate survival of Bacillus anthracis Ames, B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores after exposure to PES-Solid (a solid source of peracetic acid), including PES-Solid formulations with bacteriostatic surfactants. Spores (≥ 7 logs) were dried on seven different test materials and treated with three different PES-Solid formulations (or preneutralized controls) at room temperature for 15 min. There was either no spore survival or less than 1 log (<10 spores) of spore survival in 56 of 63 test combinations (strain, formulation and substrate). Less than 2.7 logs (<180 spores) survived in the remaining seven test combinations. The highest spore survival rates were seen on water-dispersible chemical agent resistant coating (CARC-W) and Naval ship topcoat (NTC). Electron microscopy and Coulter analysis showed that all spore structures were intact after spore inactivation with PES-Solid. Three PES-Solid formulations inactivated Bacillus spores that were dried on seven different materials. A test method was developed to show that PES-Solid formulations effectively inactivate Bacillus spores on different materials. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  2. DNA glue

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V; Astakhova, Irina V.; Malakhov, Andrei D.

    2008-01-01

    Significant alterations in thermal stability of parallel DNA triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in the structure of phenylmethylglycerol inserted as a bulge into DNA (TINA). Insertions of two ortho-TINAs as a pseudo...

  3. Hyperstretching DNA

    NARCIS (Netherlands)

    Schakenraad, Koen; Biebricher, Andreas S.; Sebregts, Maarten; Ten Bensel, Brian; Peterman, Erwin J.G.; Wuite, Gijs J L; Heller, Iddo; Storm, Cornelis; Van Der Schoot, Paul

    2017-01-01

    The three-dimensional structure of DNA is highly susceptible to changes by mechanical and biochemical cues in vivo and in vitro. In particular, large increases in base pair spacing compared to regular B-DNA are effected by mechanical (over)stretching and by intercalation of compounds that are widely

  4. DNA probes

    International Nuclear Information System (INIS)

    Castelino, J.

    1992-01-01

    The creation of DNA probes for detection of specific nucleotide segments differs from ligand detection in that it is a chemical rather than an immunological reaction. Complementary DNA or RNA is used in place of the antibody and is labelled with 32 P. So far, DNA probes have been successfully employed in the diagnosis of inherited disorders, infectious diseases, and for identification of human oncogenes. The latest approach to the diagnosis of communicable and parasitic infections is based on the use of deoxyribonucleic acid (DNA) probes. The genetic information of all cells is encoded by DNA and DNA probe approach to identification of pathogens is unique because the focus of the method is the nucleic acid content of the organism rather than the products that the nucleic acid encodes. Since every properly classified species has some unique nucleotide sequences that distinguish it from every other species, each organism's genetic composition is in essence a finger print that can be used for its identification. In addition to this specificity, DNA probes offer other advantages in that pathogens may be identified directly in clinical specimens

  5. DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Castelino, J

    1993-12-31

    The creation of DNA probes for detection of specific nucleotide segments differs from ligand detection in that it is a chemical rather than an immunological reaction. Complementary DNA or RNA is used in place of the antibody and is labelled with {sup 32}P. So far, DNA probes have been successfully employed in the diagnosis of inherited disorders, infectious diseases, and for identification of human oncogenes. The latest approach to the diagnosis of communicable and parasitic infections is based on the use of deoxyribonucleic acid (DNA) probes. The genetic information of all cells is encoded by DNA and DNA probe approach to identification of pathogens is unique because the focus of the method is the nucleic acid content of the organism rather than the products that the nucleic acid encodes. Since every properly classified species has some unique nucleotide sequences that distinguish it from every other species, each organism`s genetic composition is in essence a finger print that can be used for its identification. In addition to this specificity, DNA probes offer other advantages in that pathogens may be identified directly in clinical specimens 10 figs, 2 tabs

  6. False Negative Rates of a Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates via Real-Time PCR

    Energy Technology Data Exchange (ETDEWEB)

    Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Amidan, Brett G. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sydor, Michael A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Deatherage Kaiser, Brooke L [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2015-05-01

    Surface sampling for Bacillus anthracis spores has traditionally relied on detection via bacterial cultivation methods. Although effective, this approach does not provide the level of organism specificity that can be gained through molecular techniques. False negative rates (FNR) and limits of detection (LOD) were determined for two B. anthracis surrogates with modified rapid viability-polymerase chain reaction (mRV-PCR) following macrofoam-swab sampling. This study was conducted in parallel with a previously reported study that analyzed spores using a plate-culture method. B. anthracis Sterne (BAS) or B. atrophaeus Nakamura (BG) spores were deposited onto four surface materials (glass, stainless steel, vinyl tile, and plastic) at nine target concentrations (2 to 500 spores/coupon; 0.078 to 19.375 colony-forming units [CFU] per cm²). Mean FNR values for mRV-PCR analysis ranged from 0 to 0.917 for BAS and 0 to 0.875 for BG and increased as spore concentration decreased (over the concentrations investigated) for each surface material. FNRs based on mRV-PCR data were not statistically different for BAS and BG, but were significantly lower for glass than for vinyl tile. FNRs also tended to be lower for the mRV-PCR method compared to the culture method. The mRV-PCR LOD₉₅ was lowest for glass (0.429 CFU/cm² with BAS and 0.341 CFU/cm² with BG) and highest for vinyl tile (0.919 CFU/cm² with BAS and 0.917 CFU/cm² with BG). These mRV-PCR LOD₉₅ values were lower than the culture values (BAS: 0.678 to 1.023 CFU/cm² and BG: 0.820 to 1.489 CFU/cm²). The FNR and LOD₉₅ values reported in this work provide guidance for environmental sampling of Bacillus spores at low concentrations.

  7. Inactivation of Bacillus anthracis Spores by a Combination of Biocides and Heating under High-Temperature Short-Time Pasteurization Conditions ▿

    Science.gov (United States)

    Xu, Sa; Labuza, Theodore P.; Diez-Gonzalez, Francisco

    2008-01-01

    The milk supply is considered a primary route for a bioterrorism attack with Bacillus anthracis spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate B. anthracis spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85°C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80°C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80°C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80°C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80°C, respectively. TTI6-log of less than 20 s were also achieved at 80°C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of B. anthracis spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be used in HTST milk plants to process

  8. Inactivation of Bacillus anthracis spores by a combination of biocides and heating under high-temperature short-time pasteurization conditions.

    Science.gov (United States)

    Xu, Sa; Labuza, Theodore P; Diez-Gonzalez, Francisco

    2008-06-01

    The milk supply is considered a primary route for a bioterrorism attack with Bacillus anthracis spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate B. anthracis spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85 degrees C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80 degrees C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80 degrees C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80 degrees C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80 degrees C, respectively. TTI6-log of less than 20 s were also achieved at 80 degrees C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of B. anthracis spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be

  9. Detection of anthrax lef with DNA-based photonic crystal sensors

    Science.gov (United States)

    Zhang, Bailin; Dallo, Shatha; Peterson, Ralph; Hussain, Syed; Weitao, Tao; Ye, Jing Yong

    2011-12-01

    Bacillus anthracis has posed a threat of becoming biological weapons of mass destruction due to its virulence factors encoded by the plasmid-borne genes, such as lef for lethal factor. We report the development of a fast and sensitive anthrax DNA biosensor based on a photonic crystal structure used in a total-internal-reflection configuration. For the detection of the lef gene, a single-stranded DNA lef probe was biotinylated and immobilized onto the sensor via biotin-streptavidin interactions. A positive control, lef-com, was the complementary strand of the probe, while a negative control was an unrelated single-stranded DNA fragment from the 16S rRNA gene of Acinetobacter baumannii. After addition of the biotinylated lef probe onto the sensor, significant changes in the resonance wavelength of the sensor were observed, resulting from binding of the probe to streptavidin on the sensor. The addition of lef-com led to another significant increase as a result of hybridization between the two DNA strands. The detection sensitivity for the target DNA reached as low as 0.1 nM. In contrast, adding the unrelated DNAs did not cause an obvious shift in the resonant wavelength. These results demonstrate that detection of the anthrax lef by the photonic crystal structure in a total-internal-reflection sensor is highly specific and sensitive.

  10. DNA methylation

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Helin, Kristian

    2012-01-01

    DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent...... discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET...... proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation...

  11. DNA data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Raw DNA chromatogram data produced by the ABI 373, 377, 3130 and 3730 automated sequencing machines in ABI format. These are from fish (primarily Sebastes spp.,...

  12. DNA nanotechnology

    Science.gov (United States)

    Seeman, Nadrian C.; Sleiman, Hanadi F.

    2018-01-01

    DNA is the molecule that stores and transmits genetic information in biological systems. The field of DNA nanotechnology takes this molecule out of its biological context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnology, and has been revolutionary in our ability to control molecular self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomolecule structure determination, drug delivery and synthetic biology. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.

  13. DNA expressions - A formal notation for DNA

    NARCIS (Netherlands)

    Vliet, Rudy van

    2015-01-01

    We describe a formal notation for DNA molecules that may contain nicks and gaps. The resulting DNA expressions denote formal DNA molecules. Different DNA expressions may denote the same molecule. Such DNA expressions are called equivalent. We examine which DNA expressions are minimal, which

  14. What Is Mitochondrial DNA?

    Science.gov (United States)

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  15. Experimental Design for a Macrofoam-Swab Study Relating the Recovery Efficiency and False Negative Rate to Low Concentrations of Two Bacillus anthracis Surrogates on Four Surface Materials

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2014-12-05

    This report describes the experimental design for a laboratory study to quantify the recovery efficiencies and false negative rates of a validated, macrofoam-swab sampling method for low concentrations of Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus (BG) spores on four surface materials (stainless steel, glass, vinyl tile, plastic light cover panel). Two analytical methods (culture and polymerase chain reaction) will be used. Only one previous study has investigated how the false negative rate depends on test factors. The surrogates BAS and BG have not been tested together in the same study previously. Hence, this study will provide for completing gaps in the available information on the performance of macrofoam-swab sampling at low concentrations.

  16. Experimental Design for a Macrofoam Swab Study Relating the Recovery Efficiency and False Negative Rate to Low Concentrations of Two Bacillus anthracis Surrogates on Four Surface Materials

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F.; Hutchison, Janine R.

    2014-04-16

    This report describes the experimental design for a laboratory study to quantify the recovery efficiencies and false negative rates of a validated, macrofoam swab sampling method for low concentrations of Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus (BG) spores on four surface materials (stainless steel, glass, vinyl tile, plastic light cover panel). Two analytical methods (plating/counting and polymerase chain reaction) will be used. Only one previous study has investigated false negative as a function of affecting test factors. The surrogates BAS and BG have not been tested together in the same study previously. Hence, this study will provide for completing gaps in the available information on the performance of macrofoam swab sampling at low concentrations.

  17. Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

    Science.gov (United States)

    Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

    2014-10-01

    Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  18. Immunological correlates for protection against intranasal challenge of Bacillus anthracis spores conferred by a protective antigen-based vaccine in rabbits.

    Science.gov (United States)

    Weiss, Shay; Kobiler, David; Levy, Haim; Marcus, Hadar; Pass, Avi; Rothschild, Nili; Altboum, Zeev

    2006-01-01

    Correlates between immunological parameters and protection against Bacillus anthracis infection in animals vaccinated with protective antigen (PA)-based vaccines could provide surrogate markers to evaluate the putative protective efficiency of immunization in humans. In previous studies we demonstrated that neutralizing antibody levels serve as correlates for protection in guinea pigs (S. Reuveny et al., Infect. Immun. 69:2888-2893, 2001; H. Marcus et al., Infect. Immun. 72:3471-3477, 2004). In this study we evaluated similar correlates for protection by active and passive immunization of New Zealand White rabbits. Full immunization and partial immunization were achieved by single and multiple injections of standard and diluted doses of a PA-based vaccine. Passive immunization was carried out by injection of immune sera from rabbits vaccinated with PA-based vaccine prior to challenge with B. anthracis spores. Immunized rabbits were challenged by intranasal spore instillation with one of two virulent strains (strains Vollum and ATCC 6605). The immune competence was estimated by measuring the level of total anti-PA antibodies, the neutralizing antibody titers, and the conferred protective immunity. The results indicate that total anti-PA antibody titers greater than 1 x 10(5) conferred protection, whereas lower titers (between 10(4) and 10(5)) provided partial protection but failed to predict protection. Neutralizing antibody titers between 500 and 800 provided partial protection, while titers higher than 1,000 conferred protection. In conclusion, this study emphasizes that regardless of the immunization regimen or the time of challenge, neutralizing antibody titers are better predictors of protection than total anti-PA titers.

  19. Development of Protective Immunity in New Zealand White Rabbits Challenged with Bacillus anthracis Spores and Treated with Antibiotics and Obiltoxaximab, a Monoclonal Antibody against Protective Antigen.

    Science.gov (United States)

    Henning, Lisa N; Carpenter, Sarah; Stark, Gregory V; Serbina, Natalya V

    2018-02-01

    The recommended management of inhalational anthrax, a high-priority bioterrorist threat, includes antibiotics and antitoxins. Obiltoxaximab, a chimeric monoclonal antibody against anthrax protective antigen (PA), is licensed under the U.S. Food and Drug Administration's (FDA's) Animal Rule for the treatment of inhalational anthrax. Because of spore latency, disease reemergence after treatment cessation is a concern, and there is a need to understand the development of endogenous protective immune responses following antitoxin-containing anthrax treatment regimens. Here, acquired protective immunity was examined in New Zealand White (NZW) rabbits challenged with a targeted lethal dose of Bacillus anthracis spores and treated with antibiotics, obiltoxaximab, or a combination of both. Survivors of the primary challenge were rechallenged 9 months later and monitored for survival. Survival rates after primary and rechallenge for controls and animals treated with obiltoxaximab, levofloxacin, or a combination of both were 0, 65, 100, and 95%, and 0, 100, 95, and 89%, respectively. All surviving immune animals had circulating antibodies to PA and serum toxin-neutralizing titers prior to rechallenge. Following rechallenge, systemic bacteremia and toxemia were not detected in most animals, and the levels of circulating anti-PA IgG titers increased starting at 5 days postrechallenge. We conclude that treatment with obiltoxaximab, alone or combined with antibiotics, significantly improves the survival of rabbits that received a lethal inhalation B. anthracis spore challenge dose and does not interfere with the development of immunity. Survivors of primary challenge are protected against reexposure, have rare incidents of systemic bacteremia and toxemia, and have evidence of an anamnestic response. Copyright © 2018 Henning et al.

  20. Rapid Identification of Genetic Modifications in Bacillus anthracis Using Whole Genome Draft Sequences Generated by 454 Pyrosequencing

    Science.gov (United States)

    2010-08-25

    in honey bee colony collapse disorder. Science 318: 283–287. 39. Towner JS, Sealy TK, Khristova ML, Albarino CG, Conlan S, et al. (2008) Newly...utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel natural variations...detection assays utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel

  1. Pilot-scale crossflow-microfiltration and pasteurization to remove spores of Bacillus anthracis (Sterne) from milk.

    Science.gov (United States)

    Tomasula, P M; Mukhopadhyay, S; Datta, N; Porto-Fett, A; Call, J E; Luchansky, J B; Renye, J; Tunick, M

    2011-09-01

    High-temperature, short-time pasteurization of milk is ineffective against spore-forming bacteria such as Bacillus anthracis (BA), but is lethal to its vegetative cells. Crossflow microfiltration (MF) using ceramic membranes with a pore size of 1.4 μm has been shown to reject most microorganisms from skim milk; and, in combination with pasteurization, has been shown to extend its shelf life. The objectives of this study were to evaluate MF for its efficiency in removing spores of the attenuated Sterne strain of BA from milk; to evaluate the combined efficiency of MF using a 0.8-μm ceramic membrane, followed by pasteurization (72°C, 18.6s); and to monitor any residual BA in the permeates when stored at temperatures of 4, 10, and 25°C for up to 28 d. In each trial, 95 L of raw skim milk was inoculated with about 6.5 log(10) BA spores/mL of milk. It was then microfiltered in total recycle mode at 50°C using ceramic membranes with pore sizes of either 0.8 μm or 1.4 μm, at crossflow velocity of 6.2 m/s and transmembrane pressure of 127.6 kPa, conditions selected to exploit the selectivity of the membrane. Microfiltration using the 0.8-μm membrane removed 5.91±0.05 log(10) BA spores/mL of milk and the 1.4-μm membrane removed 4.50±0.35 log(10) BA spores/mL of milk. The 0.8-μm membrane showed efficient removal of the native microflora and both membranes showed near complete transmission of the casein proteins. Spore germination was evident in the permeates obtained at 10, 30, and 120 min of MF time (0.8-μm membrane) but when stored at 4 or 10°C, spore levels were decreased to below detection levels (≤0.3 log(10) spores/mL) by d 7 or 3 of storage, respectively. Permeates stored at 25°C showed coagulation and were not evaluated further. Pasteurization of the permeate samples immediately after MF resulted in additional spore germination that was related to the length of MF time. Pasteurized permeates obtained at 10 min of MF and stored at 4 or 10°C showed no

  2. The Poly-γ-D-Glutamic Acid Capsule of Bacillus licheniformis, a Surrogate of Bacillus anthracis Capsule Induces Interferon-Gamma Production in NK Cells through Interactions with Macrophages.

    Science.gov (United States)

    Lee, Hae-Ri; Jeon, Jun Ho; Rhie, Gi-Eun

    2017-05-28

    The poly-γ- D -glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis , provides protection of the bacterium from phagocytosis and allows its unimpeded growth in the host. We investigated crosstalk between murine natural killer (NK) cells and macrophages stimulated with the PGA capsule of Bacillus licheniformis , a surrogate of the B. anthracis capsule. PGA induced interferon-gamma production from NK cells cultured with macrophages. This effect was dependent on macrophage-derived IL-12 and cell-cell contact interaction with macrophages through NK cell receptor NKG2D and its ligand RAE-1. The results showed that PGA could enhance NK cell activation by inducing IL-12 production in macrophages and a contact-dependent crosstalk with macrophages.

  3. Effects of Intermediates between Vitamins K2 and K3 on Mammalian DNA Polymerase Inhibition and Anti-Inflammatory Activity

    Directory of Open Access Journals (Sweden)

    Takeshi Azuma

    2011-02-01

    Full Text Available Previously, we reported that vitamin K3 (VK3, but not VK1 or VK2 (=MK-4, inhibits the activity of human DNA polymerase γ (pol γ. In this study, we chemically synthesized three intermediate compounds between VK2 and VK3, namely MK-3, MK-2 and MK-1, and investigated the inhibitory effects of all five compounds on the activity of mammalian pols. Among these compounds, MK-2 was the strongest inhibitor of mammalian pols α, κ and λ, which belong to the B, Y and X families of pols, respectively; whereas VK3 was the strongest inhibitor of human pol γ, an A-family pol. MK-2 potently inhibited the activity of all animal species of pol tested, and its inhibitory effect on pol λ activity was the strongest with an IC50 value of 24.6 μM. However, MK-2 did not affect the activity of plant or prokaryotic pols, or that of other DNA metabolic enzymes such as primase of pol α, RNA polymerase, polynucleotide kinase or deoxyribonuclease I. Because we previously found a positive relationship between pol λ inhibition and anti-inflammatory action, we examined whether these compounds could inhibit inflammatory responses. Among the five compounds tested, MK-2 caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA-induced acute inflammation in mouse ear. In addition, in a cell culture system using mouse macrophages, MK-2 displayed the strongest suppression of the production of tumor necrosis factor (TNF-α induced by lipopolysaccharide (LPS. Moreover, MK-2 was found to inhibit the action of nuclear factor (NF-κB. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of MK-2 in mice led to suppression of TNF-α production in serum. In conclusion, this study has identified VK2 and VK3 intermediates, such as MK-2, that are promising anti-inflammatory candidates.

  4. Genetic evidence for the involvement of the S-layer protein gene sap and the sporulation genes spo0A, spo0B, and spo0F in Phage AP50c infection of Bacillus anthracis.

    Science.gov (United States)

    Plaut, Roger D; Beaber, John W; Zemansky, Jason; Kaur, Ajinder P; George, Matroner; Biswas, Biswajit; Henry, Matthew; Bishop-Lilly, Kimberly A; Mokashi, Vishwesh; Hannah, Ryan M; Pope, Robert K; Read, Timothy D; Stibitz, Scott; Calendar, Richard; Sozhamannan, Shanmuga

    2014-03-01

    In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on whole-genome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wild-type B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer.

  5. DNA Vaccines

    Indian Academy of Sciences (India)

    diseases. Keywords. DNA vaccine, immune response, antibodies, infectious diseases. GENERAL .... tein vaccines require expensive virus/protein purification tech- niques as ... sphere continue to remain major health hazards in developing nations. ... significance since it can be produced at a very low cost and can be stored ...

  6. DNA Investigations.

    Science.gov (United States)

    Mayo, Ellen S.; Bertino, Anthony J.

    1991-01-01

    Presents a simulation activity that allow students to work through the exercise of DNA profiling and to grapple with some analytical and ethical questions involving a couple arranging with a surrogate mother to have a baby. Can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. (MDH)

  7. DNA repair

    International Nuclear Information System (INIS)

    Setlow, R.

    1978-01-01

    Some topics discussed are as follows: difficulty in extrapolating data from E. coli to mammalian systems; mutations caused by UV-induced changes in DNA; mutants deficient in excision repair; other postreplication mechanisms; kinds of excision repair systems; detection of repair by biochemical or biophysical means; human mutants deficient in repair; mutagenic effects of UV on XP cells; and detection of UV-repair defects among XP individuals

  8. DNA repair

    International Nuclear Information System (INIS)

    Van Zeeland, A.A.

    1984-01-01

    In this chapter a series of DNA repair pathways are discussed which are available to the cell to cope with the problem of DNA damaged by chemical or physical agents. In the case of microorganisms our knowledge about the precise mechanism of each DNA repair pathway and the regulation of it has been improved considerably when mutants deficient in these repair mechanisms became available. In the case of mammalian cells in culture, until recently there were very little repair deficient mutants available, because in almost all mammalian cells in culture at least the diploid number of chromosomes is present. Therefore the frequency of repair deficient mutants in such populations is very low. Nevertheless because replica plating techniques are improving some mutants from Chinese hamsters ovary cells and L5178Y mouse lymphoma cells are now available. In the case of human cells, cultures obtained from patients with certain genetic diseases are available. A number of cells appear to be sensitive to some chemical or physical mutagens. These include cells from patients suffering from xeroderma pigmentosum, Ataxia telangiectasia, Fanconi's anemia, Cockayne's syndrome. However, only in the case of xeroderma pigmentosum cells, has the sensitivity to ultraviolet light been clearly correlated with a deficiency in excision repair of pyrimidine dimers. Furthermore the work with strains obtained from biopsies from man is difficult because these cells generally have low cloning efficiencies and also have a limited lifespan in vitro. It is therefore very important that more repair deficient mutants will become available from established cell lines from human or animal origin

  9. Recovery and identification of bacterial DNA from illicit drugs.

    Science.gov (United States)

    Cho, Kaymann T; Richardson, Michelle M; Kirkbride, K Paul; McNevin, Dennis; Nelson, Michelle; Pianca, Dennis; Roffey, Paul; Gahan, Michelle E

    2014-02-01

    Bacterial infections, including Bacillus anthracis (anthrax), are a common risk associated with illicit drug use, particularly among injecting drug users. There is, therefore, an urgent need to survey illicit drugs used for injection for the presence of bacteria and provide valuable information to health and forensic authorities. The objectives of this study were to develop a method for the extraction of bacterial DNA from illicit drugs and conduct a metagenomic survey of heroin and methamphetamine seized in the Australian Capital Territory during 2002-2011 for the presence of pathogens. Trends or patterns in drug contamination and their health implications for injecting drug users were also investigated. Methods based on the ChargeSwitch(®)gDNA mini kit (Invitrogen), QIAamp DNA extraction mini kit (QIAGEN) with and without bead-beating, and an organic phenol/chloroform extraction with ethanol precipitation were assessed for the recovery efficiency of both free and cellular bacterial DNA. Bacteria were identified using polymerase chain reaction and electrospray ionization-mass spectrometry (PCR/ESI-MS). An isopropanol pre-wash to remove traces of the drug and diluents, followed by a modified ChargeSwitch(®) method, was found to efficiently lyse cells and extract free and cellular DNA from Gram-positive and Gram-negative bacteria in heroin and methamphetamine which could then be identified by PCR/ESI-MS. Analysis of 12 heroin samples revealed the presence of DNA from species of Comamonas, Weissella, Bacillus, Streptococcus and Arthrobacter. No organisms were detected in the nine methamphetamine samples analysed. This study develops a method to extract and identify Gram-positive and Gram-negative bacteria from illicit drugs and demonstrates the presence of a range of bacterial pathogens in seized drug samples. These results will prove valuable for future work investigating trends or patterns in drug contamination and their health implications for injecting drug

  10. Structure of the Bacillus anthracis dTDP- L -rhamnose-biosynthetic enzyme glucose-1-phosphate thymidylyltransferase (RfbA)

    Energy Technology Data Exchange (ETDEWEB)

    Baumgartner, Jackson; Lee, Jesi; Halavaty, Andrei S.; Minasov, George; Anderson, Wayne F.; Kuhn, Misty L. (NWU); (SFSU)

    2017-10-30

    L-Rhamnose is a ubiquitous bacterial cell-wall component. The biosynthetic pathway for its precursor dTDP-L-rhamnose is not present in humans, which makes the enzymes of the pathway potential drug targets. In this study, the three-dimensional structure of the first protein of this pathway, glucose-1-phosphate thymidylyltransferase (RfbA), fromBacillus anthraciswas determined. In other organisms this enzyme is referred to as RmlA. RfbA was co-crystallized with the products of the enzymatic reaction, dTDP-α-D-glucose and pyrophosphate, and its structure was determined at 2.3 Å resolution. This is the first reported thymidylyltransferase structure from a Gram-positive bacterium. RfbA shares overall structural characteristics with known RmlA homologs. However, RfbA exhibits a shorter sequence at its C-terminus, which results in the absence of three α-helices involved in allosteric site formation. Consequently, RfbA was observed to exhibit a quaternary structure that is unique among currently reported glucose-1-phosphate thymidylyltransferase bacterial homologs. These structural analyses suggest that RfbA may not be allosterically regulated in some organisms and is structurally distinct from other RmlA homologs.

  11. DNA Repair Systems

    Indian Academy of Sciences (India)

    DNA molecule which makes it ideal for storage and propagation of genetic information. ... of these errors are broadly referred to as DNA repair. DNA can ... changes occur in the human genome per day. ..... nails, frequent physical and mental.

  12. Synthesis of DNA

    Science.gov (United States)

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  13. Inactivation of Vegetative Cells, but Not Spores, of Bacillus anthracis, B. cereus, and B. subtilis on Stainless Steel Surfaces Coated with an Antimicrobial Silver- and Zinc-Containing Zeolite Formulation

    Science.gov (United States)

    Galeano, Belinda; Korff, Emily; Nicholson, Wayne L.

    2003-01-01

    Stainless steel surfaces coated with paints containing a silver- and zinc-containing zeolite (AgION antimicrobial) were assayed in comparison to uncoated stainless steel for antimicrobial activity against vegetative cells and spores of three Bacillus species, namely, B. anthracis Sterne, B. cereus T, and B. subtilis 168. Under the test conditions (25°C and 80% relative humidity), the zeolite coating produced approximately 3 log10 inactivation of vegetative cells within a 5- to 24-h period, but viability of spores of the three species was not significantly affected. PMID:12839825

  14. Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

    Science.gov (United States)

    Jahouh, Farid; Hou, Shu-jie; Kováč, Pavol; Banoub, Joseph H.

    2012-01-01

    We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate–protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate–spacer–carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate. PMID:22012665

  15. Recovery Efficiency, False Negative Rate, and Limit of Detection Performance of a Validated Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Deatherage Kaiser, Brooke L [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Amidan, Brett G. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sydor, Michael A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Barrett, Christopher A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2015-03-31

    The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target amounts (2-500 spores) to positive-control plates and test coupons (2 in. × 2 in.) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest for vinyl tile (50.8% with BAS, 40.2% with BG) and the highest for glass (92.8% with BAS, 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG, with values increasing as concentration decreased in the range tested (0.078 to 19.375 CFU/cm2, where CFU denotes ‘colony forming units’). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results will be discussed in a subsequent report.

  16. Recovery Efficiency, False Negative Rate, and Limit of Detection Performance of a Validated Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Kaiser, Brooke L. D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Amidan, Brett G. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sydor, Michael A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Barrett, Christopher A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-06-16

    The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target amounts (2-500 spores) to positive-control plates and test coupons (2 in × 2 in) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest for vinyl tile (50.8% with BAS, 40.2% with BG) and the highest for glass (92.8% with BAS, 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG, with values increasing as concentration decreased in the range tested (0.078 to 19.375 CFU/cm2, where CFU denotes ‘colony forming units’). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results are discussed in a separate report.

  17. Decontamination efficacy of three commercial-off-the-shelf (COTS sporicidal disinfectants on medium-sized panels contaminated with surrogate spores of Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Jason M Edmonds

    Full Text Available In the event of a wide area release and contamination of a biological agent in an outdoor environment and to building exteriors, decontamination is likely to consume the Nation's remediation capacity, requiring years to cleanup, and leading to incalculable economic losses. This is in part due to scant body of efficacy data on surface areas larger than those studied in a typical laboratory (5×10-cm, resulting in low confidence for operational considerations in sampling and quantitative measurements of prospective technologies recruited in effective cleanup and restoration response. In addition to well-documented fumigation-based cleanup efforts, agencies responsible for mitigation of contaminated sites are exploring alternative methods for decontamination including combinations of disposal of contaminated items, source reduction by vacuuming, mechanical scrubbing, and low-technology alternatives such as pH-adjusted bleach pressure wash. If proven effective, a pressure wash-based removal of Bacillus anthracis spores from building surfaces with readily available equipment will significantly increase the readiness of Federal agencies to meet the daunting challenge of restoration and cleanup effort following a wide-area biological release. In this inter-agency study, the efficacy of commercial-of-the-shelf sporicidal disinfectants applied using backpack sprayers was evaluated in decontamination of spores on the surfaces of medium-sized (∼1.2 m2 panels of steel, pressure-treated (PT lumber, and brick veneer. Of the three disinfectants, pH-amended bleach, Peridox, and CASCAD evaluated; CASCAD was found to be the most effective in decontamination of spores from all three panel surface types.

  18. Mapping the epitopes of a neutralizing antibody fragment directed against the lethal factor of Bacillus anthracis and cross-reacting with the homologous edema factor.

    Directory of Open Access Journals (Sweden)

    Philippe Thullier

    Full Text Available The lethal toxin (LT of Bacillus anthracis, composed of the protective antigen (PA and the lethal factor (LF, plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF to form the edema toxin (ET, which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236, of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260 was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.

  19. Bacterial DNA of Ocean and Land on the Surface of the International Space Station.

    Science.gov (United States)

    Grebennikova, Tatiana

    A.V. Syroeshkin2, T.V. Grebennikova1, E.V. Shubralova3, V.A. Shuvalov3, O.S. Tsygankov4, V.B. Lapshin2 1D. I. Ivanovsky Virology Institute, Moscow, Russia 2 Academician E. K. Fedorov Institute of Applied Geophysics, Moscow, Russia 3S.P. Korolev Rocket and Space Corporation «Energia» Korolev, Russia 4Central Research Institute of Machine Building, Korolev, Russia Existence of biological molecules as markers of microorganisms in the space environment has always attracted attention of researchers. There is great attention to the search for extraterrestrial life forms [Nicholson W.L. 2009, Kawaguchi Y. et al 2013], and as well as the coping mechanisms of living organisms in the interplanetary space [Hotchin J. et al 1965, Baranov V.M. 2009, Horneck G. et al 2010]. Experiments on American and Japanese segments of the International Space Station (ISS) over the different nature of resistance during prolonged stay in space were conducted [Scalzi G et al 2012, Wassmann M. et al 2012]. As a result of these experiments confirmed the possibility of preserving the viability of organisms in an open space for a long time. Consequence, became interested in the transfer of living matter from the stratosphere to near-Earth space [Smith D.J. 2013]. We hypothesized that viable forms, or at least, intact DNA can be transferred to the orbit of the ISS with the ascending branch of the global electric circuit. Samples of cosmic dust collected from the surface of the window of the ISS during the exit of an astronaut in space. Samples (washes with material of tampons and tampons) which were in vacuo, were analyzed for the presence of bacterial DNA by nested PCR using primers specific DNA genus Mycobacterium, the DNA of the strain of the genus Bacillus anthracis and DNA encoding the bacterial 16S ribosomal RNA after transportation of the samples to Earth. The results of amplification, followed by sequencing and phylogenetic analysis showed the presence in samples of cosmic dust DNA

  20. Inhibition of the adenylyl cyclase toxin, edema factor, from Bacillus anthracis by a series of 18 mono- and bis-(M)ANT-substituted nucleoside 5'-triphosphates.

    Science.gov (United States)

    Taha, Hesham; Dove, Stefan; Geduhn, Jens; König, Burkhard; Shen, Yuequan; Tang, Wei-Jen; Seifert, Roland

    2012-01-01

    Bacillus anthracis causes anthrax disease and exerts its deleterious effects by the release of three exotoxins, i.e. lethal factor, protective antigen and edema factor (EF), a highly active calmodulin-dependent adenylyl cyclase (AC). Conventional antibiotic treatment is ineffective against either toxaemia or antibiotic-resistant strains. Thus, more effective drugs for anthrax treatment are needed. Our previous studies showed that EF is differentially inhibited by various purine and pyrimidine nucleotides modified with N-methylanthraniloyl (MANT)- or anthraniloyl (ANT) groups at the 2'(3')-O-ribosyl position, with the unique preference for the base cytosine (Taha et al., Mol Pharmacol 75:693 (2009)). MANT-CTP was the most potent EF inhibitor (K (i), 100 nM) among 16 compounds studied. Here, we examined the interaction of EF with a series of 18 2',3'-O-mono- and bis-(M)ANT-substituted nucleotides, recently shown to be very potent inhibitors of the AC toxin from Bordetella pertussis, CyaA (Geduhn et al., J Pharmacol Exp Ther 336:104 (2011)). We analysed purified EF and EF mutants in radiometric AC assays and in fluorescence spectroscopy studies and conducted molecular modelling studies. Bis-MANT nucleotides inhibited EF competitively. Propyl-ANT-ATP was the most potent EF inhibitor (K (i), 80 nM). In contrast to the observations made for CyaA, introduction of a second (M)ANT-group decreased rather than increased inhibitor potency at EF. Activation of EF by calmodulin resulted in effective fluorescence resonance energy transfer (FRET) from tryptophan and tyrosine residues located in the vicinity of the catalytic site to bis-MANT-ATP, but FRET to bis-MANT-CTP was only small. Mutations N583Q, K353A and K353R differentially altered the inhibitory potencies of bis-MANT-ATP and bis-MANT-CTP. The nucleotide binding site of EF accommodates bulky bis-(M)ANT-substituted purine and pyrimidine nucleotides, but the fit is suboptimal compared to CyaA. These data provide a basis

  1. DNA preservation in silk.

    Science.gov (United States)

    Liu, Yawen; Zheng, Zhaozhu; Gong, He; Liu, Meng; Guo, Shaozhe; Li, Gang; Wang, Xiaoqin; Kaplan, David L

    2017-06-27

    The structure of DNA is susceptible to alterations at high temperature and on changing pH, irradiation and exposure to DNase. Options to protect and preserve DNA during storage are important for applications in genetic diagnosis, identity authentication, drug development and bioresearch. In the present study, the stability of total DNA purified from human dermal fibroblast cells, as well as that of plasmid DNA, was studied in silk protein materials. The DNA/silk mixtures were stabilized on filter paper (silk/DNA + filter) or filter paper pre-coated with silk and treated with methanol (silk/DNA + PT-filter) as a route to practical utility. After air-drying and water extraction, 50-70% of the DNA and silk could be retrieved and showed a single band on electrophoretic gels. 6% silk/DNA + PT-filter samples provided improved stability in comparison with 3% silk/DNA + filter samples and DNA + filter samples for DNA preservation, with ∼40% of the band intensity remaining at 37 °C after 40 days and ∼10% after exposure to UV light for 10 hours. Quantitative analysis using the PicoGreen assay confirmed the results. The use of Tris/borate/EDTA (TBE) buffer enhanced the preservation and/or extraction of the DNA. The DNA extracted after storage maintained integrity and function based on serving as a functional template for PCR amplification of the gene for zinc finger protein 750 (ZNF750) and for transgene expression of red fluorescence protein (dsRed) in HEK293 cells. The high molecular weight and high content of a crystalline beta-sheet structure formed on the coated surfaces likely accounted for the preservation effects observed for the silk/DNA + PT-filter samples. Although similar preservation effects were also obtained for lyophilized silk/DNA samples, the rapid and simple processing available with the silk-DNA-filter membrane system makes it appealing for future applications.

  2. Force induced DNA melting

    International Nuclear Information System (INIS)

    Santosh, Mogurampelly; Maiti, Prabal K

    2009-01-01

    When pulled along the axis, double-strand DNA undergoes a large conformational change and elongates by roughly twice its initial contour length at a pulling force of about 70 pN. The transition to this highly overstretched form of DNA is very cooperative. Applying a force perpendicular to the DNA axis (unzipping), double-strand DNA can also be separated into two single-stranded DNA, this being a fundamental process in DNA replication. We study the DNA overstretching and unzipping transition using fully atomistic molecular dynamics (MD) simulations and argue that the conformational changes of double-strand DNA associated with either of the above mentioned processes can be viewed as force induced DNA melting. As the force at one end of the DNA is increased the DNA starts melting abruptly/smoothly above a critical force depending on the pulling direction. The critical force f m , at which DNA melts completely decreases as the temperature of the system is increased. The melting force in the case of unzipping is smaller compared to the melting force when the DNA is pulled along the helical axis. In the case of melting through unzipping, the double-strand separation has jumps which correspond to the different energy minima arising due to sequence of different base pairs. The fraction of Watson-Crick base pair hydrogen bond breaking as a function of force does not show smooth and continuous behavior and consists of plateaus followed by sharp jumps.

  3. DNA damage and autophagy

    International Nuclear Information System (INIS)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Panayiotidis, Mihalis I.; Franco, Rodrigo

    2011-01-01

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  4. DNA Open states and DNA hydratation

    International Nuclear Information System (INIS)

    Lema-Larre, B. de; Martin-Landrove, M

    1995-01-01

    It is a very well-known fact that an protonic exchange exists among natural DNA filaments and synthetic polynucleotides with the solvent (1--2). The existence of DNA open states, that is to say states for which the interior of the DNA molecule is exposed to the external environment, it has been demonstrated by means of proton-deuterium exchange (3). This work has carried out experiments measuring the dispersion of the traverse relaxation rate (4), as a pulsation rate function in a Carr-Purcell-Meiboom-Gill (CPMG) pulses sequence rate, to determine changes in the moist layer of the DNA molecule. The experiments were carried out under different experimental conditions in order to vary the probability that open states occurs, such as temperature or the exposure to electromagnetic fields. Some theoretical models were supposed to adjust the experimental results including those related to DNA non linear dynamic [es

  5. Immunoassay of DNA damage

    International Nuclear Information System (INIS)

    Gasparro, F.P.; Santella, R.M.

    1988-01-01

    The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA). (author)

  6. Immunoassay of DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Gasparro, F P; Santella, R M

    1988-09-01

    The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA).

  7. DNA computing models

    CERN Document Server

    Ignatova, Zoya; Zimmermann, Karl-Heinz

    2008-01-01

    In this excellent text, the reader is given a comprehensive introduction to the field of DNA computing. The book emphasizes computational methods to tackle central problems of DNA computing, such as controlling living cells, building patterns, and generating nanomachines.

  8. DNA tagged microparticles

    Science.gov (United States)

    Farquar, George Roy; Leif, Roald N; Wheeler, Elizabeth

    2015-05-05

    A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

  9. Modeling DNA Replication.

    Science.gov (United States)

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  10. Conjugative plasmid pAW63 brings new insights into the genesis of the Bacillus anthracis virulence plasmid pXO2 and of the Bacillus thuringiensis plasmid pBT9727

    Directory of Open Access Journals (Sweden)

    Mahillon Jacques

    2005-07-01

    Full Text Available Abstract Background Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis belong to the genetically close-knit Bacillus cereus sensu lato group, a family of rod-shaped Gram-positive bacteria. pAW63 is the first conjugative plasmid from the B. cereus group to be completely sequenced. Results The 71,777 bp nucleotide sequence of pAW63 reveals a modular structure, including a 42 kb tra region encoding homologs of the Type IV secretion systems components VirB11, VirB4 and VirD4, as well as homologs of Gram-positive conjugation genes from Enterococcus, Lactococcus, Listeria, Streptococcus and Staphylococcus species. It also firmly establishes the existence of a common backbone between pAW63, pXO2 from Bacillus anthracis and pBT9727 from the pathogenic Bacillus thuringiensis serovar konkukian strain 97-27. The alignment of these three plasmids highlights the presence of well conserved segments, in contrast to distinct regions of high sequence plasticity. The study of their specific differences has provided a three-point reference framework that can be exploited to formulate solid hypotheses concerning the functionalities and the molecular evolution of these three closely related plasmids. This has provided insight into the chronology of their divergence, and led to the discovery of two Type II introns on pAW63, matching copies of the mobile element IS231L in different loci of pXO2 and pBT9727, and the identification on pXO2 of a 37 kb pathogenicity island (PAI containing the anthrax capsule genes. Conclusion The complete sequence determination of pAW63 has led to a functional map of the plasmid yielding insights into its conjugative apparatus, which includes T4SS-like components, as well as its resemblance to other large plasmids of Gram-positive bacteria. Of particular interest is the extensive homology shared between pAW63 and pXO2, the second virulence plasmid of B. anthracis, as well as pBT9727 from the pathogenic strain B. thuringiensis

  11. DNA: Structure and function

    DEFF Research Database (Denmark)

    Sinden, Richard R.; E. Pearson, Christopher; N. Potaman, Vladimir

    1998-01-01

    This chapter discusses the structure and function of DNA. DNA occupies a critical role in cells, because it is the source of all intrinsic genetic information. Chemically, DNA is a very stable molecule, a characteristic important for a macromolecule that may have to persist in an intact form...

  12. Fast phylogenetic DNA barcoding

    DEFF Research Database (Denmark)

    Terkelsen, Kasper Munch; Boomsma, Wouter Krogh; Willerslev, Eske

    2008-01-01

    We present a heuristic approach to the DNA assignment problem based on phylogenetic inferences using constrained neighbour joining and non-parametric bootstrapping. We show that this method performs as well as the more computationally intensive full Bayesian approach in an analysis of 500 insect...... DNA sequences obtained from GenBank. We also analyse a previously published dataset of environmental DNA sequences from soil from New Zealand and Siberia, and use these data to illustrate the fact that statistical approaches to the DNA assignment problem allow for more appropriate criteria...... for determining the taxonomic level at which a particular DNA sequence can be assigned....

  13. Radiation and DNA

    Energy Technology Data Exchange (ETDEWEB)

    Riabchenko, N I

    1979-01-01

    Consideration is given to the effects of ionizing radiation on the structure of DNA. Physical and chemical methods of determining radiation damage to the primary (polynucleotide chain and nitrogenous base) and secondary (helical) structure of DNA are discussed, and the effects of ionizing radiation on deoxyribonucleoprotein complexes are considered. The radiolysis of DNA in vitro and in bacterial and mammalian cells is examined and cellular mechanisms for the repair of radiation-damaged DNA are considered, taking into account single-strand and double-strand breaks, gamma-radiation damage and deoxyribonucleoprotein-membrane complex damage. Postradiation DNA degradation in bacteria and lymphatic cells is also discussed.

  14. DNA-Mediated Electrochemistry

    Science.gov (United States)

    Gorodetsky, Alon A.; Buzzeo, Marisa C.

    2009-01-01

    The base pair stack of DNA has been demonstrated as a medium for long range charge transport chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to structural perturbations in the base pair stack as occur with lesions, single base mismatches, and protein binding. We have exploited this sensitivity for the development of reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers. Here we discuss the characteristic features, applications, and advantages of DNA-mediated electrochemistry. PMID:18980370

  15. DNA fragmentation in spermatozoa

    DEFF Research Database (Denmark)

    Rex, A S; Aagaard, J.; Fedder, J

    2017-01-01

    Sperm DNA Fragmentation has been extensively studied for more than a decade. In the 1940s the uniqueness of the spermatozoa protein complex which stabilizes the DNA was discovered. In the fifties and sixties, the association between unstable chromatin structure and subfertility was investigated....... In the seventies, the impact of induced DNA damage was investigated. In the 1980s the concept of sperm DNA fragmentation as related to infertility was introduced as well as the first DNA fragmentation test: the Sperm Chromatin Structure Assay (SCSA). The terminal deoxynucleotidyl transferase nick end labelling...... (TUNEL) test followed by others was introduced in the nineties. The association between DNA fragmentation in spermatozoa and pregnancy loss has been extensively investigated spurring the need for a therapeutic tool for these patients. This gave rise to an increased interest in the aetiology of DNA damage...

  16. Biophysics of DNA

    CERN Document Server

    Vologodskii, Alexander

    2015-01-01

    Surveying the last sixty years of research, this book describes the physical properties of DNA in the context of its biological functioning. It is designed to enable both students and researchers of molecular biology, biochemistry and physics to better understand the biophysics of DNA, addressing key questions and facilitating further research. The chapters integrate theoretical and experimental approaches, emphasising throughout the importance of a quantitative knowledge of physical properties in building and analysing models of DNA functioning. For example, the book shows how the relationship between DNA mechanical properties and the sequence specificity of DNA-protein binding can be analyzed quantitatively by using our current knowledge of the physical and structural properties of DNA. Theoretical models and experimental methods in the field are critically considered to enable the reader to engage effectively with the current scientific literature on the physical properties of DNA.

  17. Use of DNA Microarrays to Identify Diagnostic Signature Transcriptional Profiles for Host Responses to Infectious Agents

    National Research Council Canada - National Science Library

    Ellner, J. J; Connell, N. D; Gallagher, G; Raveche, E

    2005-01-01

    Infections by agents of bioterrorism, especially bacterial agents such as Bacillus anthracis and Yersinia pestis present their initial symptoms in a way that does not reveal their identity or permit rapid diagnosis...

  18. Beyond DNA repair: DNA-PK function in cancer

    OpenAIRE

    Goodwin, Jonathan F.; Knudsen, Karen E.

    2014-01-01

    The DNA-dependent protein kinase (DNA-PK) is a pivotal component of the DNA repair machinery that governs the response to DNA damage, serving to maintain genome integrity. However, the DNA-PK kinase component was initially isolated with transcriptional complexes, and recent findings have illuminated the impact of DNA-PK-mediated transcriptional regulation on tumor progression and therapeutic response. DNA-PK expression has also been correlated with poor outcome in selected tumor types, furthe...

  19. DNA topology and transcription

    Science.gov (United States)

    Kouzine, Fedor; Levens, David; Baranello, Laura

    2014-01-01

    Chromatin is a complex assembly that compacts DNA inside the nucleus while providing the necessary level of accessibility to regulatory factors conscripted by cellular signaling systems. In this superstructure, DNA is the subject of mechanical forces applied by variety of molecular motors. Rather than being a rigid stick, DNA possesses dynamic structural variability that could be harnessed during critical steps of genome functioning. The strong relationship between DNA structure and key genomic processes necessitates the study of physical constrains acting on the double helix. Here we provide insight into the source, dynamics, and biology of DNA topological domains in the eukaryotic cells and summarize their possible involvement in gene transcription. We emphasize recent studies that might inspire and impact future experiments on the involvement of DNA topology in cellular functions. PMID:24755522

  20. Eukaryotic DNA Replication Fork.

    Science.gov (United States)

    Burgers, Peter M J; Kunkel, Thomas A

    2017-06-20

    This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

  1. DNA-based machines.

    Science.gov (United States)

    Wang, Fuan; Willner, Bilha; Willner, Itamar

    2014-01-01

    The base sequence in nucleic acids encodes substantial structural and functional information into the biopolymer. This encoded information provides the basis for the tailoring and assembly of DNA machines. A DNA machine is defined as a molecular device that exhibits the following fundamental features. (1) It performs a fuel-driven mechanical process that mimics macroscopic machines. (2) The mechanical process requires an energy input, "fuel." (3) The mechanical operation is accompanied by an energy consumption process that leads to "waste products." (4) The cyclic operation of the DNA devices, involves the use of "fuel" and "anti-fuel" ingredients. A variety of DNA-based machines are described, including the construction of "tweezers," "walkers," "robots," "cranes," "transporters," "springs," "gears," and interlocked cyclic DNA structures acting as reconfigurable catenanes, rotaxanes, and rotors. Different "fuels", such as nucleic acid strands, pH (H⁺/OH⁻), metal ions, and light, are used to trigger the mechanical functions of the DNA devices. The operation of the devices in solution and on surfaces is described, and a variety of optical, electrical, and photoelectrochemical methods to follow the operations of the DNA machines are presented. We further address the possible applications of DNA machines and the future perspectives of molecular DNA devices. These include the application of DNA machines as functional structures for the construction of logic gates and computing, for the programmed organization of metallic nanoparticle structures and the control of plasmonic properties, and for controlling chemical transformations by DNA machines. We further discuss the future applications of DNA machines for intracellular sensing, controlling intracellular metabolic pathways, and the use of the functional nanostructures for drug delivery and medical applications.

  2. DNA repair and cancer

    International Nuclear Information System (INIS)

    Rathore, Shakuntla; Joshi, Pankaj Kumar; Gaur, Sudha

    2012-01-01

    DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecule that encode it's genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many one million individual molecular lesions per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions include potentially harmful mutation in cell's genome which affect the survival of it's daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. Inherited mutation that affect DNA repair genes are strongly associated with high cancer risks in humans. Hereditary non polyposis colorectal cancer (HNPCC) is strongly associated with specific mutation in the DNA mismatch repair pathway. BRCA1, BRCA2 two famous mutation conferring a hugely increased risk of breast cancer on carrier, are both associated with a large number of DNA repair pathway, especially NHEJ and homologous recombination. Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing most typically cancer cells are preferentially affected. The side effect is that other non-cancerous but rapidly dividing cells such as stem cells in the bone marrow are also affected. Modern cancer treatment attempt to localize the DNA damage to cells and tissue only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body). (author)

  3. Nonisotopic DNA probe techniques

    National Research Council Canada - National Science Library

    Kricka, Larry J

    1992-01-01

    The objective of this book is to bring together descriptions of the principal nonisotopic methods for DNA hybridization assays, together with experimental details of the methods, including labelling...

  4. DNA replication and cancer

    DEFF Research Database (Denmark)

    Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

    2016-01-01

    A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways...... causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy....

  5. Forensic DNA testing.

    Science.gov (United States)

    Butler, John M

    2011-12-01

    Forensic DNA testing has a number of applications, including parentage testing, identifying human remains from natural or man-made disasters or terrorist attacks, and solving crimes. This article provides background information followed by an overview of the process of forensic DNA testing, including sample collection, DNA extraction, PCR amplification, short tandem repeat (STR) allele separation and sizing, typing and profile interpretation, statistical analysis, and quality assurance. The article concludes with discussions of possible problems with the data and other forensic DNA testing techniques.

  6. Structure of DNA toroids and electrostatic attraction of DNA duplexes

    International Nuclear Information System (INIS)

    Cherstvy, A G

    2005-01-01

    DNA-DNA electrostatic attraction is considered as the driving force for the formation of DNA toroids in the presence of DNA condensing cations. This attraction comes from the DNA helical charge distribution and favours hexagonal toroidal cross-sections. The latter is in agreement with recent cryo-electron microscopy studies on DNA condensed with cobalt hexammine. We treat the DNA-DNA interactions within the modern theory of electrostatic interaction between helical macromolecules. The size and thickness of the toroids is calculated within a simple model; other models of stability of DNA toroids are discussed and compared

  7. Colorimetric and dynamic light scattering detection of DNA sequences by using positively charged gold nanospheres: a comparative study with gold nanorods

    Science.gov (United States)

    Pylaev, T. E.; Khanadeev, V. A.; Khlebtsov, B. N.; Dykman, L. A.; Bogatyrev, V. A.; Khlebtsov, N. G.

    2011-07-01

    We introduce a new genosensing approach employing CTAB (cetyltrimethylammonium bromide)-coated positively charged colloidal gold nanoparticles (GNPs) to detect target DNA sequences by using absorption spectroscopy and dynamic light scattering. The approach is compared with a previously reported method employing unmodified CTAB-coated gold nanorods (GNRs). Both approaches are based on the observation that whereas the addition of probe and target ssDNA to CTAB-coated particles results in particle aggregation, no aggregation is observed after addition of probe and nontarget DNA sequences. Our goal was to compare the feasibility and sensitivity of both methods. A 21-mer ssDNA from the human immunodeficiency virus type 1 HIV-1 U5 long terminal repeat (LTR) sequence and a 23-mer ssDNA from the Bacillus anthracis cryptic protein and protective antigen precursor (pagA) genes were used as ssDNA models. In the case of GNRs, unexpectedly, the colorimetric test failed with perfect cigar-like particles but could be performed with dumbbell and dog-bone rods. By contrast, our approach with cationic CTAB-coated GNPs is easy to implement and possesses excellent feasibility with retention of comparable sensitivity—a 0.1 nM concentration of target cDNA can be detected with the naked eye and 10 pM by dynamic light scattering (DLS) measurements. The specificity of our method is illustrated by successful DLS detection of one-three base mismatches in cDNA sequences for both DNA models. These results suggest that the cationic GNPs and DLS can be used for genosensing under optimal DNA hybridization conditions without any chemical modifications of the particle surface with ssDNA molecules and signal amplification. Finally, we discuss a more than two-three-order difference in the reported estimations of the detection sensitivity of colorimetric methods (0.1 to 10-100 pM) to show that the existing aggregation models are inconsistent with the detection limits of about 0.1-1 pM DNA and that

  8. Extended DNA Tile Actuators

    DEFF Research Database (Denmark)

    Kristiansen, Martin; Kryger, Mille; Zhang, Zhao

    2012-01-01

    A dynamic linear DNA tile actuator is expanded to three new structures of higher complexity. The original DNA actuator was constructed from a central roller strand which hybridizes with two piston strands by forming two half-crossover junctions. A linear expansion of the actuator is obtained...

  9. Dna fingerprinting - review paper

    OpenAIRE

    Blundell, Renald

    2006-01-01

    Before the Polymerase Chain Reaction (PCR) was established, DNA fingerprinting technology has relied for years on Restriction Fragment Length Polymorphism (RFLP) and Variable Number of Tandom Repeats (VNTR) analysis, a very efficient technique but quite laborious and not suitable for high throughput mapping. Since its, development, PCR has provided a new and powerful tool for DNA fingerprinting.

  10. DNA Repair Systems

    Indian Academy of Sciences (India)

    Thanks to the pioneering research work of Lindahl, Sancar, Modrich and their colleagues, we now have an holistic awareness of how DNA damage occurs and how the damage is rectified in bacteria as well as in higher organisms including human beings. A comprehensive understanding of DNA repair has proven crucial ...

  11. DNA repair genes

    International Nuclear Information System (INIS)

    Morimyo, Mitsuoki

    1995-01-01

    Fission yeast S. pombe is assumed to be a good model for cloning of human DNA repair genes, because human gene is normally expressed in S. pombe and has a very similar protein sequence to yeast protein. We have tried to elucidate the DNA repair mechanisms of S. pombe as a model system for those of mammals. (J.P.N.)

  12. DNA-cell conjugates

    Science.gov (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2018-05-15

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  13. Characterization of muntjac DNA

    International Nuclear Information System (INIS)

    Davis, R.C.

    1981-01-01

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange

  14. DNA-cell conjugates

    Science.gov (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  15. Characterization of muntjac DNA

    Energy Technology Data Exchange (ETDEWEB)

    Davis, R.C.

    1981-05-27

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange.

  16. Whose DNA is this?

    DEFF Research Database (Denmark)

    Taroni, Franco; Biedermann, Alex; Vuille, Joëlle

    2013-01-01

    This communication seeks to draw the attention of researchers and practitioners dealing with forensic DNA profiling analyses to the following question: is a scientist's report, offering support to a hypothesis according to which a particular individual is the source of DNA detected during...... evoked during the international conference "The hidden side of DNA profiles. Artifacts, errors and uncertain evidence" held in Rome (April 27th to 28th, 2012). Indeed, despite the fact that this conference brought together some of the world's leading forensic DNA specialists, it appeared clearly...... talk considerably different languages. It thus is fundamental to address this issue of communication about results of forensic DNA analyses, and open a dialogue with practicing non-scientists at large who need to make meaningful use of scientific results to approach and help solve judicial cases...

  17. DNA repair protocols

    DEFF Research Database (Denmark)

    Bjergbæk, Lotte

    In its 3rd edition, this Methods in Molecular Biology(TM) book covers the eukaryotic response to genomic insult including advanced protocols and standard techniques in the field of DNA repair. Offers expert guidance for DNA repair, recombination, and replication. Current knowledge of the mechanisms...... that regulate DNA repair has grown significantly over the past years with technology advances such as RNA interference, advanced proteomics and microscopy as well as high throughput screens. The third edition of DNA Repair Protocols covers various aspects of the eukaryotic response to genomic insult including...... recent advanced protocols as well as standard techniques used in the field of DNA repair. Both mammalian and non-mammalian model organisms are covered in the book, and many of the techniques can be applied with only minor modifications to other systems than the one described. Written in the highly...

  18. Racemic DNA crystallography.

    Science.gov (United States)

    Mandal, Pradeep K; Collie, Gavin W; Kauffmann, Brice; Huc, Ivan

    2014-12-22

    Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of L- and D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propensity of racemic DNA mixtures to form racemic crystals. We describe racemic crystal structures of various DNA sequences and folded conformations, including duplexes, quadruplexes, and a four-way junction, showing that the advantages of racemic crystallography should extend to DNA. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Elaborated Action of the Human Primosome

    Directory of Open Access Journals (Sweden)

    Andrey G. Baranovskiy

    2017-02-01

    Full Text Available The human primosome is a 340-kilodalton complex of primase (DNA-dependent RNA polymerase and DNA polymerase α, which initiates genome replication by synthesizing chimeric RNA-DNA primers for DNA polymerases δ and ϵ. Accumulated biochemical and structural data reveal the complex mechanism of concerted primer synthesis by two catalytic centers. First, primase generates an RNA primer through three steps: initiation, consisting of dinucleotide synthesis from two nucleotide triphosphates; elongation, resulting in dinucleotide extension; and termination, owing to primase inhibition by a mature 9-mer primer. Then Polα, which works equally well on DNA:RNA and DNA:DNA double helices, intramolecularly catches the template primed by a 9mer RNA and extends the primer with dNTPs. All primosome transactions are highly coordinated by autoregulation through the alternating activation/inhibition of the catalytic centers. This coordination is mediated by the small C-terminal domain of the primase accessory subunit, which forms a tight complex with the template:primer, shuttles between the primase and DNA polymerase active sites, and determines their access to the substrate.

  20. Nanostructures via DNA scaffold metallization

    OpenAIRE

    Ning, C.; Zinchenko, A.; Baigl, D.; Pyshkina, O.; Sergeyev, V.; Endo, Kazunaka; Yoshikawa, K.

    2005-01-01

    The critical role of polymers in process of noble metals nanostructures formation is well known, however, the use of DNA chain template in this process is yet largely unknown. In this study we demonstrate different ways of silver deposition on DNA template and report the influence of silver nanostructures formation on DNA conformational state. Metallization of DNA chain proceeds by two different scenarios depending on DNA conformation. If DNA chain is unfolded (elongated) chain, silver reduct...

  1. DNA damage and polyploidization.

    Science.gov (United States)

    Chow, Jeremy; Poon, Randy Y C

    2010-01-01

    A growing body of evidence indicates that polyploidization triggers chromosomal instability and contributes to tumorigenesis. DNA damage is increasingly being recognized for its roles in promoting polyploidization. Although elegant mechanisms known as the DNA damage checkpoints are responsible for halting the cell cycle after DNA damage, agents that uncouple the checkpoints can induce unscheduled entry into mitosis. Likewise, defects of the checkpoints in several disorders permit mitotic entry even in the presence of DNA damage. Forcing cells with damaged DNA into mitosis causes severe chromosome segregation defects, including lagging chromosomes, chromosomal fragments and chromosomal bridges. The presence of these lesions in the cleavage plane is believed to abort cytokinesis. It is postulated that if cytokinesis failure is coupled with defects of the p53-dependent postmitotic checkpoint pathway, cells can enter S phase and become polyploids. Progress in the past several years has unraveled some of the underlying principles of these pathways and underscored the important role of DNA damage in polyploidization. Furthermore, polyploidization per se may also be an important determinant of sensitivity to DNA damage, thereby may offer an opportunity for novel therapies.

  2. Quantitive DNA Fiber Mapping

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  3. Regulating DNA Self-assembly by DNA-Surface Interactions.

    Science.gov (United States)

    Liu, Longfei; Li, Yulin; Wang, Yong; Zheng, Jianwei; Mao, Chengde

    2017-12-14

    DNA self-assembly provides a powerful approach for preparation of nanostructures. It is often studied in bulk solution and involves only DNA-DNA interactions. When confined to surfaces, DNA-surface interactions become an additional, important factor to DNA self-assembly. However, the way in which DNA-surface interactions influence DNA self-assembly is not well studied. In this study, we showed that weak DNA-DNA interactions could be stabilized by DNA-surface interactions to allow large DNA nanostructures to form. In addition, the assembly can be conducted isothermally at room temperature in as little as 5 seconds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  5. "Artifactual" arsenate DNA

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2012-01-01

    The recent claim by Wolfe-Simon et al. that the Halomonas bacterial strain GFAJ-1 when grown in arsenate-containing medium with limiting phosphate is able to substitute phosphate with arsenate in biomolecules including nucleic acids and in particular DNA(1) arose much skepticism, primarily due...... to the very limited chemical stability of arsenate esters (see ref. 2 and references therein). A major part of the criticisms was concerned with the insufficient (bio)chemical evidence in the Wolfe-Simon study for the actual chemical incorporation of arsenate in DNA (and/or RNA). Redfield et al. now present...... evidence that the identification of arsenate DNA was artifactual....

  6. The differential effects of heat-shocking on the viability of spores from Bacillus anthracis, Bacillus subtilis, and Clostridium sporogenes after treatment with peracetic acid- and glutaraldehyde-based disinfectants.

    Science.gov (United States)

    March, Jordon K; Pratt, Michael D; Lowe, Chinn-Woan; Cohen, Marissa N; Satterfield, Benjamin A; Schaalje, Bruce; O'Neill, Kim L; Robison, Richard A

    2015-10-01

    This study investigated (1) the susceptibility of Bacillus anthracis (Ames strain), Bacillus subtilis (ATCC 19659), and Clostridium sporogenes (ATCC 3584) spores to commercially available peracetic acid (PAA)- and glutaraldehyde (GA)-based disinfectants, (2) the effects that heat-shocking spores after treatment with these disinfectants has on spore recovery, and (3) the timing of heat-shocking after disinfectant treatment that promotes the optimal recovery of spores deposited on carriers. Suspension tests were used to obtain inactivation kinetics for the disinfectants against three spore types. The effects of heat-shocking spores after disinfectant treatment were also determined. Generalized linear mixed models were used to estimate 6-log reduction times for each spore type, disinfectant, and heat treatment combination. Reduction times were compared statistically using the delta method. Carrier tests were performed according to AOAC Official Method 966.04 and a modified version that employed immediate heat-shocking after disinfectant treatment. Carrier test results were analyzed using Fisher's exact test. PAA-based disinfectants had significantly shorter 6-log reduction times than the GA-based disinfectant. Heat-shocking B. anthracis spores after PAA treatment resulted in significantly shorter 6-log reduction times. Conversely, heat-shocking B. subtilis spores after PAA treatment resulted in significantly longer 6-log reduction times. Significant interactions were also observed between spore type, disinfectant, and heat treatment combinations. Immediately heat-shocking spore carriers after disinfectant treatment produced greater spore recovery. Sporicidal activities of disinfectants were not consistent across spore species. The effects of heat-shocking spores after disinfectant treatment were dependent on both disinfectant and spore species. Caution must be used when extrapolating sporicidal data of disinfectants from one spore species to another. Heat

  7. DNA from keratinous tissue

    DEFF Research Database (Denmark)

    Bengtsson, Camilla F.; Olsen, Maja E.; Brandt, Luise Ørsted

    2011-01-01

    Keratinous tissues such as nail, hair, horn, scales and feather have been used as a source of DNA for over 20 years. Particular benefits of such tissues include the ease with which they can be sampled, the relative stability of DNA in such tissues once sampled, and, in the context of ancient...... genetic analyses, the fact that sampling generally causes minimal visual damage to valuable specimens. Even when freshly sampled, however, the DNA quantity and quality in the fully keratinized parts of such tissues is extremely poor in comparison to other tissues such as blood and muscle – although little...... systematic research has been undertaken to characterize how such degradation may relate to sample source. In this review paper we present the current understanding of the quality and limitations of DNA in two key keratinous tissues, nail and hair. The findings indicate that although some fragments of nuclear...

  8. DNA fusion gene vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Bassi, Maria Rosaria; Thomsen, Allan Randrup

    2010-01-01

    DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion...... of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular...... with viral-vectored vaccines, various synergistic components may need to be incorporated into DNA vaccines. From the perspective of the future clinical use of DNA vaccines, it has been suggested that antigen presentation should be improved and cytokine coadministration attempted. However, even...

  9. DNA Sampling Hook

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The DNA Sampling Hook is a significant improvement on a method of obtaining a tissue sample from a live fish in situ from an aquatic environment. A tissue sample...

  10. Retroviral DNA Integration

    Science.gov (United States)

    2016-01-01

    The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications. PMID:27198982

  11. DNA damage and carcinogenesis

    International Nuclear Information System (INIS)

    Stelow, R.B.

    1980-01-01

    Although cancer may arise as a result of many different types of molecular changes, there is little reason to doubt that changes to DNA are one of the more important ones in cancer initiation. Although DNA repair mechanisms seem able to eliminate a very large fraction of deleterious changes to DNA, we not only have little insight into the molecular mechanisms involved in such repair, but have a negligible amount of information to permit us to estimate the shape of dose response relations at low doses. The case of skin cancer is a special one, in that the average population is exposed to sufficient solar uv so that the effects of small increments in uv dose may be estimated. An approximate 85% reduction in DNA repair increases skin cancer incidence 10 4 fold

  12. DNA-Origami

    DEFF Research Database (Denmark)

    Voigt, Niels Vinther; Tørring, Thomas; Gothelf, Kurt Vesterager

    2010-01-01

    DNA-nanostrukturer giver nye muligheder for studier af individuelle molekyler. Ved at udnytte DNAs unikke selvsamlende egenskaber kan man designe systemer, hvorpå der kan studeres kemiske reaktioner, fluoroforer og biiomolekyler på enkeltmolekyle-niveau....

  13. DNA Microarray Technology

    Science.gov (United States)

    Skip to main content DNA Microarray Technology Enter Search Term(s): Español Research Funding An Overview Bioinformatics Current Grants Education and Training Funding Extramural Research News Features Funding Divisions Funding ...

  14. DNA sequencing conference, 2

    Energy Technology Data Exchange (ETDEWEB)

    Cook-Deegan, R.M. [Georgetown Univ., Kennedy Inst. of Ethics, Washington, DC (United States); Venter, J.C. [National Inst. of Neurological Disorders and Strokes, Bethesda, MD (United States); Gilbert, W. [Harvard Univ., Cambridge, MA (United States); Mulligan, J. [Stanford Univ., CA (United States); Mansfield, B.K. [Oak Ridge National Lab., TN (United States)

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  15. Close encounters with DNA

    Science.gov (United States)

    Maffeo, C.; Yoo, J.; Comer, J.; Wells, D. B.; Luan, B.; Aksimentiev, A.

    2014-01-01

    Over the past ten years, the all-atom molecular dynamics method has grown in the scale of both systems and processes amenable to it and in its ability to make quantitative predictions about the behavior of experimental systems. The field of computational DNA research is no exception, witnessing a dramatic increase in the size of systems simulated with atomic resolution, the duration of individual simulations and the realism of the simulation outcomes. In this topical review, we describe the hallmark physical properties of DNA from the perspective of all-atom simulations. We demonstrate the amazing ability of such simulations to reveal the microscopic physical origins of experimentally observed phenomena and we review the frustrating limitations associated with imperfections of present atomic force fields and inadequate sampling. The review is focused on the following four physical properties of DNA: effective electric charge, response to an external mechanical force, interaction with other DNA molecules and behavior in an external electric field. PMID:25238560

  16. Gomphid DNA sequence data

    Data.gov (United States)

    U.S. Environmental Protection Agency — DNA sequence data for several genetic loci. This dataset is not publicly accessible because: It's already publicly available on GenBank. It can be accessed through...

  17. HPV DNA test

    Science.gov (United States)

    ... test; Cancer of cervix - HPV DNA test References Hacker NF. Cervical dysplasia and cancer. In: Hacker NF, Gambone JC, Hobel CJ, eds. Hacker and Moore's Essentials of Obstetrics and Gynecology . 6th ...

  18. Close encounters with DNA.

    Science.gov (United States)

    Maffeo, C; Yoo, J; Comer, J; Wells, D B; Luan, B; Aksimentiev, A

    2014-10-15

    Over the past ten years, the all-atom molecular dynamics method has grown in the scale of both systems and processes amenable to it and in its ability to make quantitative predictions about the behavior of experimental systems. The field of computational DNA research is no exception, witnessing a dramatic increase in the size of systems simulated with atomic resolution, the duration of individual simulations and the realism of the simulation outcomes. In this topical review, we describe the hallmark physical properties of DNA from the perspective of all-atom simulations. We demonstrate the amazing ability of such simulations to reveal the microscopic physical origins of experimentally observed phenomena. We also discuss the frustrating limitations associated with imperfections of present atomic force fields and inadequate sampling. The review is focused on the following four physical properties of DNA: effective electric charge, response to an external mechanical force, interaction with other DNA molecules and behavior in an external electric field.

  19. FBI's DNA analysis program

    Science.gov (United States)

    Brown, John R.

    1994-03-01

    Forensic DNA profiling technology is a significant law enforcement tool due to its superior discriminating power. Applying the principles of population genetics to the DNA profile obtained in violent crime investigations results in low frequency of occurrence estimates for the DNA profile. These estimates often range from a frequency of occurrence of 1 in 50 unrelated individuals to 1 in a million unrelated individuals or even smaller. It is this power to discriminate among individuals in the population that has propelled forensic DNA technology to the forefront of forensic testing in violent crime cases. Not only is the technology extremely powerful in including or excluding a criminal suspect as the perpetrator, but it also gives rise to the potential of identifying criminal suspects in cases where the investigators of unknown suspect cases have exhausted all other available leads.

  20. Making DNA Fingerprints.

    Science.gov (United States)

    Nunley, Kathie F.

    1996-01-01

    Presents an activity to simulate electrophoresis using everyday items. Uses adding machine paper to construct a set of DNA fingerprints that can be used to solve crime cases designed by students in any biology class. (JRH)

  1. Radiation damage in DNA

    International Nuclear Information System (INIS)

    Lafleur, V.

    1978-01-01

    A number of experiments are described with the purpose to obtain a better insight in the chemical nature and the biological significance of radiation-induced damage in DNA, with some emphasis on the significance of alkali-labile sites. It is shown that not only reactions of OH radicals but also of H radicals introduce breaks and other inactivating damage in single-standed phiX174 DNA. It is found that phosphate buffer is very suitable for the study of the reactions of H radicals with DNA, as the H 2 PO 4 - ions convert the hydrated electrons into H radicals. The hydrated electron, which does react with DNA, does not cause a detectable inactivation. (Auth.)

  2. DNA to DNA transcription might exist in eukaryotic cells

    OpenAIRE

    Li, Gao-De

    2016-01-01

    Till now, in biological sciences, the term, transcription, mainly refers to DNA to RNA transcription. But our recently published experimental findings obtained from Plasmodium falciparum strongly suggest the existence of DNA to DNA transcription in the genome of eukaryotic cells, which could shed some light on the functions of certain noncoding DNA in the human and other eukaryotic genomes.

  3. Patterning nanocrystals using DNA

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Shara Carol [Univ. of California, Berkeley, CA (United States)

    2003-01-01

    One of the goals of nanotechnology is to enable programmed self-assembly of patterns made of various materials with nanometer-sized control. This dissertation describes the results of experiments templating arrangements of gold and semiconductor nanocrystals using 2'-deoxyribonucleic acid (DNA). Previously, simple DNA-templated linear arrangements of two and three nanocrystals structures have been made.[1] Here, we have sought to assemble larger and more complex nanostructures. Gold-DNA conjugates with 50 to 100 bases self-assembled into planned arrangements using strands of DNA containing complementary base sequences. We used two methods to increase the complexity of the arrangements: using branched synthetic doublers within the DNA covalent backbone to create discrete nanocrystal groupings, and incorporating the nanocrystals into a previously developed DNA lattice structure [2][3] that self-assembles from tiles made of DNA double-crossover molecules to create ordered nanoparticle arrays. In the first project, the introduction of a covalently-branched synthetic doubler reagent into the backbone of DNA strands created a branched DNA ''trimer.'' This DNA trimer templated various structures that contained groupings of three and four gold nanoparticles, giving promising, but inconclusive transmission electron microscopy (TEM) results. Due to the presence of a variety of possible structures in the reaction mixtures, and due to the difficulty of isolating the desired structures, the TEM and gel electrophoresis results for larger structures having four particles, and for structures containing both 5 and 10 nm gold nanoparticles were inconclusive. Better results may come from using optical detection methods, or from improved sample preparation. In the second project, we worked toward making two-dimensional ordered arrays of nanocrystals. We replicated and improved upon previous results for making DNA lattices, increasing the size of the lattices

  4. Das DNA-Puzzle

    Science.gov (United States)

    Kirchner, Stefan

    Im Jahre 1953 wurde von James Watson und Francis Crick erstmalig der strukturelle Aufbau der sogenannten DNA (Desoxyribonukleinsäure) beschrieben, welche das Erbgut jedes Lebewesens enthält. Der wesentliche Teil des Erbguts wird dabei durch eine sehr lange Folge der vier Basen Adenin (A), Cytosin (C), Guanin (G) und Thymin (T) codiert. Seit einigen Jahren ist es möglich, die Folge der vier Basen zu einer gegebenen DNA zu bestimmen. Biologen bezeichnen diesen Vorgang als Sequenzierung.

  5. PDA: Pooled DNA analyzer

    Directory of Open Access Journals (Sweden)

    Lin Chin-Yu

    2006-04-01

    Full Text Available Abstract Background Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer, to analyze pooled DNA data. Results We develop the software, PDA, for the analysis of pooled-DNA data. PDA is originally implemented with the MATLAB® language, but it can also be executed on a Windows system without installing the MATLAB®. PDA provides estimates of the coefficient of preferential amplification and allele frequency. PDA considers an extended single-point association test, which can compare allele frequencies between two DNA pools constructed under different experimental conditions. Moreover, PDA also provides novel chromosome-wide multipoint association tests based on p-value combinations and a sliding-window concept. This new multipoint testing procedure overcomes a computational bottleneck of conventional haplotype-oriented multipoint methods in DNA pooling analyses and can handle data sets having a large pool size and/or large numbers of polymorphic markers. All of the PDA functions are illustrated in the four bona fide examples. Conclusion PDA is simple to operate and does not require that users have a strong statistical background. The software is available at http://www.ibms.sinica.edu.tw/%7Ecsjfann/first%20flow/pda.htm.

  6. Racemic DNA Crystallography

    OpenAIRE

    Mandal , Pradeep K.; Collie , Gavin W.; Kauffmann , Brice; Huc , Ivan

    2014-01-01

    International audience; Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of Land D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propens...

  7. Celebrating DNA's Repair Crew.

    Science.gov (United States)

    Kunkel, Thomas A

    2015-12-03

    This year, the Nobel Prize in Chemistry has been awarded to Tomas Lindahl, Aziz Sancar, and Paul Modrich for their seminal studies of the mechanisms by which cells from bacteria to man repair DNA damage that is generated by normal cellular metabolism and stress from the environment. These studies beautifully illustrate the remarkable power of DNA repair to influence life from evolution through disease susceptibility. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Introduction to DNA methods

    International Nuclear Information System (INIS)

    Delincee, H.

    1991-01-01

    The purpose of this session is to discuss the various possibilities for detecting modifications in DNA after irradiation and whether these changes can be utilized as an indicator for the irradiation treatment of foods. The requirement to be fulfilled is that the method be able to distinguish irradiated food without the presence of a control sample, thus the measured response after irradiation must be large enough to supersede background levels from other treatments. Much work has been performed on the effects of radiation on DNA, particularly due to its importance in radiation biology. The main lesions of DNA as a result of irradiation are base damage, damage of the sugar moiety, single strand and double strand breaks. Crosslinking between bases also occurs, e.g. production of thymine dimers, or between DNA and protein. A valuable review on how to utilize these DNA changes for detection purposes has already appeared. Tables 1, 2 and 3 list the proposed methods of detecting changes in irradiated DNA, some identified products as examples for a possible irradiation indicator, in the case of immunoassay the substance used as antigen, and some selected literature references. In this short review, it is not intended to provide a complete literature survey

  9. Variations in brain DNA

    Directory of Open Access Journals (Sweden)

    Jesus eAvila

    2014-11-01

    Full Text Available It is assumed that DNA sequences are conserved in the diverse cell types present in a multicellular organism like the human being. Thus, in order to compare the sequences in the genome of DNA from different individuals, nucleic acid is commonly isolated from a single tissue. In this regard, blood cells are widely used for this purpose because of their availability. Thus blood DNA has been used to study genetic familiar diseases that affect other tissues and organs, such as the liver, heart, and brain. While this approach is valid for the identification of familial diseases in which mutations are present in parental germinal cells and, therefore, in all the cells of a given organism, it is not suitable to identify sporadic diseases in which mutations might occur in specific somatic cells. This review addresses somatic DNA variations in different tissues or cells (mainly in the brain of single individuals and discusses whether the dogma of DNA invariance between cell types is indeed correct. We will also discuss how single nucleotide somatic variations arise, focusing on the presence of specific DNA mutations in the brain.

  10. Blood extracellular DNA after irradiation

    International Nuclear Information System (INIS)

    Vladimirov, V.G.; Tishchenko, L.I.; Surkova, E.A.; Vasil'eva, I.N.

    1993-01-01

    It has been shown that blood extracellular DNA of irradiated rats largely consists of the low-molecular DNA and its oligomers. Molecular masses of oligomers are multiple to molecular mass of monomer fragment with nucleosome size. The low-molecular DNA has linear form. The average content of GC-pairs in low-molecular DNA is higher than in total rat's DNA (48.5% against 41.5%). The low-molecular DNA is a part of complex containing RNA, acidic proteins and lipids. It is assumed that the formation of low-molecular DNA is a result of Ca/Mg - dependent nuclear endonuclease action

  11. Evaluation of the ability of N-terminal fragment of lethal factor of Bacillus anthracis for delivery of Mycobacterium T cell antigen ESAT-6 into cytosol of antigen presenting cells to elicit effective cytotoxic T lymphocyte response

    International Nuclear Information System (INIS)

    Chandra, Subhash; Kaur, Manpreet; Midha, Shuchi; Bhatnagar, Rakesh; Banerjee-Bhatnagar, Nirupama

    2006-01-01

    We report the ability of N-terminal fragment of lethal factor of Bacillus anthracis to deliver genetically fused ESAT-6 (early secretory antigen target), a potent T cell antigen of Mycobacterium tuberculosis, into cytosol to elicit Cytotoxic T lymphocyte (CTL) response. In vitro Th1 cytokines data and CTL assay proved that efficient delivery of LFn.ESAT-6 occurs in cytosol, in the presence of protective antigen (PA), and leads to generation of effective CTL response. Since CTL response is essential for protection against intracellular pathogens and, it is well known that only single T cell epitope or single antigenic protein is not sufficient to elicit protective CTL response due to variation or polymorphism in MHC-I alleles among the individuals, we suggest that as a fusion protein LFn can be used to deliver multiepitopes of T cells or multiproteins which can generate effective CTLs against intracellular pathogens like M. tuberculosis. It can be used to enhance the protective efficacy of BCG vaccine

  12. DNA Knots: Theory and Experiments

    Science.gov (United States)

    Sumners, D. W.

    Cellular DNA is a long, thread-like molecule with remarkably complex topology. Enzymes that manipulate the geometry and topology of cellular DNA perform many vital cellular processes (including segregation of daughter chromosomes, gene regulation, DNA repair, and generation of antibody diversity). Some enzymes pass DNA through itself via enzyme-bridged transient breaks in the DNA; other enzymes break the DNA apart and reconnect it to different ends. In the topological approach to enzymology, circular DNA is incubated with an enzyme, producing an enzyme signature in the form of DNA knots and links. By observing the changes in DNA geometry (supercoiling) and topology (knotting and linking) due to enzyme action, the enzyme binding and mechanism can often be characterized. This paper will discuss some personal research history, and the tangle model for the analysis of site-specific recombination experiments on circular DNA.

  13. Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription.

    Science.gov (United States)

    Gerasimova, N S; Pestov, N A; Kulaeva, O I; Clark, D J; Studitsky, V M

    2016-05-26

    RNA polymerase II (Pol II) transcription through chromatin is accompanied by formation of small intranucleosomal DNA loops. Pol II captured within a small loop drives accumulation of DNA supercoiling, facilitating further transcription. DNA breaks relieve supercoiling and induce Pol II arrest, allowing detection of DNA damage hidden in chromatin structure.

  14. DNA replication stress restricts ribosomal DNA copy number

    Science.gov (United States)

    Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

    2017-01-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

  15. DNA replication stress restricts ribosomal DNA copy number.

    Science.gov (United States)

    Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

    2017-09-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  16. DNA replication stress restricts ribosomal DNA copy number.

    Directory of Open Access Journals (Sweden)

    Devika Salim

    2017-09-01

    Full Text Available Ribosomal RNAs (rRNAs in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  17. DNA repair synthesis in human fibroblasts requires DNA polymerase delta

    International Nuclear Information System (INIS)

    Nishida, C.; Reinhard, P.; Linn, S.

    1988-01-01

    When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone

  18. Principles of DNA architectonics: design of DNA-based nanoobjects

    International Nuclear Information System (INIS)

    Vinogradova, O A; Pyshnyi, D V

    2012-01-01

    The methods of preparation of monomeric DNA blocks that serve as key building units for the construction of complex DNA objects are described. Examples are given of the formation of DNA blocks based on native and modified oligonucleotide components using hydrogen bonding and nucleic acid-specific types of bonding and also some affinity interactions with RNA, proteins, ligands. The static discrete and periodic two- and three-dimensional DNA objects reported to date are described systematically. Methods used to prove the structures of DNA objects and the prospects for practical application of nanostructures based on DNA and its analogues in biology, medicine and biophysics are considered. The bibliography includes 195 references.

  19. DNA Topoisomerases in Transcription

    DEFF Research Database (Denmark)

    Rødgaard, Morten Terpager

    2015-01-01

    This Ph.D. thesis summarizes the main results of my studies on the interplay between DNA topoisomerases and transcription. The work was performed from 2011 to 2015 at Aarhus University in the Laboratory of Genome Research, and was supervised by associate professor Anni H. Andersen. Most of the ex......This Ph.D. thesis summarizes the main results of my studies on the interplay between DNA topoisomerases and transcription. The work was performed from 2011 to 2015 at Aarhus University in the Laboratory of Genome Research, and was supervised by associate professor Anni H. Andersen. Most...... topoisomerase-DNA cleavage complex. The second study is an investigation of how topoisomerases influence gene regulation by keeping the genome in an optimal topological state....

  20. Duplication in DNA Sequences

    Science.gov (United States)

    Ito, Masami; Kari, Lila; Kincaid, Zachary; Seki, Shinnosuke

    The duplication and repeat-deletion operations are the basis of a formal language theoretic model of errors that can occur during DNA replication. During DNA replication, subsequences of a strand of DNA may be copied several times (resulting in duplications) or skipped (resulting in repeat-deletions). As formal language operations, iterated duplication and repeat-deletion of words and languages have been well studied in the literature. However, little is known about single-step duplications and repeat-deletions. In this paper, we investigate several properties of these operations, including closure properties of language families in the Chomsky hierarchy and equations involving these operations. We also make progress toward a characterization of regular languages that are generated by duplicating a regular language.

  1. DNA methylation in obesity

    Directory of Open Access Journals (Sweden)

    Małgorzata Pokrywka

    2014-11-01

    Full Text Available The number of overweight and obese people is increasing at an alarming rate, especially in the developed and developing countries. Obesity is a major risk factor for diabetes, cardiovascular disease, and cancer, and in consequence for premature death. The development of obesity results from the interplay of both genetic and environmental factors, which include sedentary life style and abnormal eating habits. In the past few years a number of events accompanying obesity, affecting expression of genes which are not directly connected with the DNA base sequence (e.g. epigenetic changes, have been described. Epigenetic processes include DNA methylation, histone modifications such as acetylation, methylation, phosphorylation, ubiquitination, and sumoylation, as well as non-coding micro-RNA (miRNA synthesis. In this review, the known changes in the profile of DNA methylation as a factor affecting obesity and its complications are described.

  2. Repeated DNA sequences in fungi

    Energy Technology Data Exchange (ETDEWEB)

    Dutta, S K

    1974-11-01

    Several fungal species, representatives of all broad groups like basidiomycetes, ascomycetes and phycomycetes, were examined for the nature of repeated DNA sequences by DNA:DNA reassociation studies using hydroxyapatite chromatography. All of the fungal species tested contained 10 to 20 percent repeated DNA sequences. There are approximately 100 to 110 copies of repeated DNA sequences of approximately 4 x 10/sup 7/ daltons piece size of each. Repeated DNA sequence homoduplexes showed on average 5/sup 0/C difference of T/sub e/50 (temperature at which 50 percent duplexes dissociate) values from the corresponding homoduplexes of unfractionated whole DNA. It is suggested that a part of repetitive sequences in fungi constitutes mitochondrial DNA and a part of it constitutes nuclear DNA. (auth)

  3. DNA Nanotechnology for Cancer Therapy

    Science.gov (United States)

    Kumar, Vinit; Palazzolo, Stefano; Bayda, Samer; Corona, Giuseppe; Toffoli, Giuseppe; Rizzolio, Flavio

    2016-01-01

    DNA nanotechnology is an emerging and exciting field, and represents a forefront frontier for the biomedical field. The specificity of the interactions between complementary base pairs makes DNA an incredible building material for programmable and very versatile two- and three-dimensional nanostructures called DNA origami. Here, we analyze the DNA origami and DNA-based nanostructures as a drug delivery system. Besides their physical-chemical nature, we dissect the critical factors such as stability, loading capability, release and immunocompatibility, which mainly limit in vivo applications. Special attention was dedicated to highlighting the boundaries to be overcome to bring DNA nanostructures closer to the bedside of patients. PMID:27022418

  4. DNA profiling of trace DNA recovered from bedding.

    Science.gov (United States)

    Petricevic, Susan F; Bright, Jo-Anne; Cockerton, Sarah L

    2006-05-25

    Trace DNA is often detected on handled items and worn clothing examined in forensic laboratories. In this study, the potential transfer of trace DNA to bedding by normal contact, when an individual sleeps in a bed, is examined. Volunteers slept one night on a new, lower bed sheet in their own bed and one night in a bed foreign to them. Samples from the sheets were collected and analysed by DNA profiling. The results indicate that the DNA profile of an individual can be obtained from bedding after one night of sleeping in a bed. The DNA profile of the owner of the bed could also be detected in the foreign bed experiments. Since mixed DNA profiles can be obtained from trace DNA on bedding, caution should be exercised when drawing conclusions from DNA profiling results obtained from such samples. This transfer may have important repercussions in sexual assault investigations.

  5. DNA tagged microparticles

    Science.gov (United States)

    Farquar, George R.; Leif, Roald N.; Wheeler, Elizabeth

    2016-03-22

    In one embodiment, a product includes a plurality of particles, each particle including: a carrier that includes a non-toxic material; and at least one DNA barcode coupled to the carrier, where the particles each have a diameter in a range from about 1 nanometer to about 100 microns.

  6. The dynamic interplay between DNA topoisomerases and DNA topology.

    Science.gov (United States)

    Seol, Yeonee; Neuman, Keir C

    2016-11-01

    Topological properties of DNA influence its structure and biochemical interactions. Within the cell, DNA topology is constantly in flux. Transcription and other essential processes, including DNA replication and repair, not only alter the topology of the genome but also introduce additional complications associated with DNA knotting and catenation. These topological perturbations are counteracted by the action of topoisomerases, a specialized class of highly conserved and essential enzymes that actively regulate the topological state of the genome. This dynamic interplay among DNA topology, DNA processing enzymes, and DNA topoisomerases is a pervasive factor that influences DNA metabolism in vivo. Building on the extensive structural and biochemical characterization over the past four decades that has established the fundamental mechanistic basis of topoisomerase activity, scientists have begun to explore the unique roles played by DNA topology in modulating and influencing the activity of topoisomerases. In this review we survey established and emerging DNA topology-dependent protein-DNA interactions with a focus on in vitro measurements of the dynamic interplay between DNA topology and topoisomerase activity.

  7. Conformation-dependent DNA attraction

    Science.gov (United States)

    Li, Weifeng; Nordenskiöld, Lars; Zhou, Ruhong; Mu, Yuguang

    2014-05-01

    Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg2+ ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg2+ or Na+, benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg2+ bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription.Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by

  8. Melting curves of gammairradiated DNA

    International Nuclear Information System (INIS)

    Hofer, H.; Altmann, H.; Kehrer, M.

    1978-08-01

    Melting curves of gammairradiated DNA and data derived of them, are reported. The diminished stability is explained by basedestruction. DNA denatures completely at room temperature, if at least every fifth basepair is broken or weakened by irradiation. (author)

  9. An Introduction to DNA Fingerprinting.

    Science.gov (United States)

    Hepfer, Carol Ely; And Others

    1993-01-01

    Provides background information on DNA fingerprinting, and describes exercises for introducing general biology students at the high school or college level to the methodology and applications of DNA fingerprinting. (PR)

  10. Esitleti kakskeelset luulekogu "Luule DNA"

    Index Scriptorium Estoniae

    2007-01-01

    Magrelli, Valerio. Luule DNA = Il DNA della poesia / tõlkinud [ja saatesõna:] Maarja Kangro ja Kalju Kruusa. Tallinn : Koma, 2006. Sisaldab autori teksti. Esitlus 24. jaan. Kirjanike majas Tallinnas

  11. Conformation-dependent DNA attraction.

    Science.gov (United States)

    Li, Weifeng; Nordenskiöld, Lars; Zhou, Ruhong; Mu, Yuguang

    2014-06-21

    Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg(2+) ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg(2+) or Na(+), benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg(2+) bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription.

  12. Alterations of ultraviolet irradiated DNA

    International Nuclear Information System (INIS)

    Davila, C.; Garces, F.

    1980-01-01

    Thymine dimers production has been studied in several DNA- 3 H irradiated at various wave lenght of U.V. Light. The influence of dimers on the hydrodynamic and optic properties, thermal structural stability and transformant capacity of DNA have been studied too. At last the recognition and excision of dimers by the DNA-UV-Endonuclease and DNA-Polimerase-I was also studied. (author)

  13. Surface-Layer (S-Layer) Proteins Sap and EA1 Govern the Binding of the S-Layer-Associated Protein BslO at the Cell Septa of Bacillus anthracis

    Science.gov (United States)

    Kern, Valerie J.; Kern, Justin W.; Theriot, Julie A.; Schneewind, Olaf

    2012-01-01

    The Gram-positive pathogen Bacillus anthracis contains 24 genes whose products harbor the structurally conserved surface-layer (S-layer) homology (SLH) domain. Proteins endowed with the SLH domain associate with the secondary cell wall polysaccharide (SCWP) following secretion. Two such proteins, Sap and EA1, have the unique ability to self-assemble into a paracrystalline layer on the surface of bacilli and form S layers. Other SLH domain proteins can also be found within the S layer and have been designated Bacillus S-layer-associated protein (BSLs). While both S-layer proteins and BSLs bind the same SCWP, their deposition on the cell surface is not random. For example, BslO is targeted to septal peptidoglycan zones, where it catalyzes the separation of daughter cells. Here we show that an insertional lesion in the sap structural gene results in elongated chains of bacilli, as observed with a bslO mutant. The chain length of the sap mutant can be reduced by the addition of purified BslO in the culture medium. This complementation in trans can be explained by an increased deposition of BslO onto the surface of sap mutant bacilli that extends beyond chain septa. Using fluorescence microscopy, we observed that the Sap S layer does not overlap the EA1 S layer and slowly yields to the EA1 S layer in a growth-phase-dependent manner. Although present all over bacilli, Sap S-layer patches are not observed at septa. Thus, we propose that the dynamic Sap/EA1 S-layer coverage of the envelope restricts the deposition of BslO to the SCWP at septal rings. PMID:22609927

  14. Analogies and surprising differences between recombinant nitric oxide synthase-like proteins from Staphylococcus aureus and Bacillus anthracis in their interactions with l-arginine analogs and iron ligands.

    Science.gov (United States)

    Salard, Isabelle; Mercey, Emilie; Rekka, Eleni; Boucher, Jean-Luc; Nioche, Pierre; Mikula, Ivan; Martasek, Pavel; Raman, C S; Mansuy, Daniel

    2006-12-01

    Genome sequencing has recently shown the presence of genes coding for NO-synthase (NOS)-like proteins in bacteria. The roles of these proteins remain unclear. The interactions of a series of l-arginine (l-arg) analogs and iron ligands with two recombinant NOS-like proteins from Staphylococcus aureus (saNOS) and Bacillus anthracis (baNOS) have been studied by UV-visible spectroscopy. SaNOS and baNOS in their ferric native state, as well as their complexes with l-arg analogs and with various ligands, exhibit spectral characteristics highly similar to the corresponding complexes of heme-thiolate proteins such as cytochromes P450 and NOSs. However, saNOS greatly differs from baNOS at the level of three main properties: (i) native saNOS mainly exists under an hexacoordinated low-spin ferric state whereas native baNOS is mainly high-spin, (ii) the addition of tetrahydrobiopterin (H4B) or H4B analogs leads to an increase of the affinity of l-arg for saNOS but not for baNOS, and (iii) saNOS Fe(II), contrary to baNOS, binds relatively bulky ligands such as nitrosoalkanes and tert-butylisocyanide. Thus, saNOS exhibits properties very similar to those of the oxygenase domain of inducible NOS (iNOS(oxy)) not containing H4B, as expected for a NOSoxy-like protein that does not contain H4B. By contrast, the properties of baNOS which look like those of H4B-containing iNOS(oxy) are unexpected for a NOS-like protein not containing H4B. The origin of these surprising properties of baNOS remains to be determined.

  15. Interfacing DNA nanodevices with biology

    DEFF Research Database (Denmark)

    Vinther, Mathias; Kjems, Jørgen

    2016-01-01

    in biology and biomedicine acting as a molecular ‘nanorobot’ or smart drug interacting with the cellular machinery. In this review, we will explore and examine the perspective of DNA nanotechnology for such use. We summarize which requirements DNA nanostructures must fulfil to function in cellular...... environments and inside living organisms. In addition, we highlight recent advances in interfacing DNA nanostructures with biology....

  16. Human Genome Research: Decoding DNA

    Science.gov (United States)

    dropdown arrow Site Map A-Z Index Menu Synopsis Human Genome Research: Decoding DNA Resources with of the DNA double helix during April 2003. James D. Watson, Francis Crick, and Maurice Wilkins were company Celera announced the completion of a "working draft" reference DNA sequence of the human

  17. My journey to DNA repair.

    Science.gov (United States)

    Lindahl, Tomas

    2013-02-01

    I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of cancer therapy, inherited human genetic disorders and ancient DNA. I initially measured DNA decay, including rates of base loss and cytosine deamination. I have discovered several important DNA repair proteins and determined their mechanisms of action. The discovery of uracil-DNA glycosylase defined a new category of repair enzymes with each specialized for different types of DNA damage. The base excision repair pathway was first reconstituted with human proteins in my group. Cell-free analysis for mammalian nucleotide excision repair of DNA was also developed in my laboratory. I found multiple distinct DNA ligases in mammalian cells, and led the first genetic and biochemical work on DNA ligases I, III and IV. I discovered the mammalian exonucleases DNase III (TREX1) and IV (FEN1). Interestingly, expression of TREX1 was altered in some human autoimmune diseases. I also showed that the mutagenic DNA adduct O(6)-methylguanine (O(6)mG) is repaired without removing the guanine from DNA, identifying a surprising mechanism by which the methyl group is transferred to a residue in the repair protein itself. A further novel process of DNA repair discovered by my research group is the action of AlkB as an iron-dependent enzyme carrying out oxidative demethylation. Copyright © 2013. Production and hosting by Elsevier Ltd.

  18. DNA Origami-Graphene Hybrid Nanopore for DNA Detection.

    Science.gov (United States)

    Barati Farimani, Amir; Dibaeinia, Payam; Aluru, Narayana R

    2017-01-11

    DNA origami nanostructures can be used to functionalize solid-state nanopores for single molecule studies. In this study, we characterized a nanopore in a DNA origami-graphene heterostructure for DNA detection. The DNA origami nanopore is functionalized with a specific nucleotide type at the edge of the pore. Using extensive molecular dynamics (MD) simulations, we computed and analyzed the ionic conductivity of nanopores in heterostructures carpeted with one or two layers of DNA origami on graphene. We demonstrate that a nanopore in DNA origami-graphene gives rise to distinguishable dwell times for the four DNA base types, whereas for a nanopore in bare graphene, the dwell time is almost the same for all types of bases. The specific interactions (hydrogen bonds) between DNA origami and the translocating DNA strand yield different residence times and ionic currents. We also conclude that the speed of DNA translocation decreases due to the friction between the dangling bases at the pore mouth and the sequencing DNA strands.

  19. Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

    Directory of Open Access Journals (Sweden)

    Nynne Sharma

    2013-01-01

    Full Text Available DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system.

  20. DNA-DNA hybridization determined in micro-wells using covalent attachment of DNA

    DEFF Research Database (Denmark)

    Christensen, H.; Angen, Øystein; Mutters, R.

    2000-01-01

    The present study was aimed at reducing the time and labour used to perform DNA-DNA hybridizations for classification of bacteria at the species level. A micro-well-format DNA hybridization method was developed and validated. DNA extractions were performed by a small-scale method and DNA...... was sheared mechanically into fragments of between 400 and 700 bases. The hybridization conditions were calibrated according to DNA similarities obtained by the spectrophotometric method using strains within the family Pasteurellaceae, Optimal conditions were obtained with 300 ng DNA added per well and bound...... by covalent attachment to NucleoLink. Hybridization was performed with 500 ng DNA, 5% (w/w) of which was labelled with photo-activatable biotin (competitive hybridization) for 2.5 h at 65 degrees C in 2 x SSC followed by stringent washing with 2 x SSC at the same temperature. The criteria for acceptance...

  1. Dine marker har DNA

    DEFF Research Database (Denmark)

    Eckholdt, Annette; Winding, Anne; Krogh, Paul Henning

    2017-01-01

    Ordet "biodiversitet" og at det er noget, vi skal have mere af, nævnes hyppigt. Men hvad er biodiversitet, og hvordan måles det? Agrologisk har bedt et par eksperter fra Aarhus Universitet forklare, hvordan et DNA-aftryk af jord og vand kan erstatte optællinger i felten og sige noget om biodivers......Ordet "biodiversitet" og at det er noget, vi skal have mere af, nævnes hyppigt. Men hvad er biodiversitet, og hvordan måles det? Agrologisk har bedt et par eksperter fra Aarhus Universitet forklare, hvordan et DNA-aftryk af jord og vand kan erstatte optællinger i felten og sige noget om...

  2. Fleet DNA (Presentation)

    Energy Technology Data Exchange (ETDEWEB)

    Walkokwicz, K.; Duran, A.

    2014-06-01

    The Fleet DNA project objectives include capturing and quantifying drive cycle and technology variation for the multitude of medium- and heavy-duty vocations; providing a common data storage warehouse for medium- and heavy-duty vehicle fleet data across DOE activities and laboratories; and integrating existing DOE tools, models, and analyses to provide data-driven decision making capabilities. Fleet DNA advantages include: for Government - providing in-use data for standard drive cycle development, R&D, tech targets, and rule making; for OEMs - real-world usage datasets provide concrete examples of customer use profiles; for fleets - vocational datasets help illustrate how to maximize return on technology investments; for Funding Agencies - ways are revealed to optimize the impact of financial incentive offers; and for researchers -a data source is provided for modeling and simulation.

  3. Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage Phi29.

    Science.gov (United States)

    Keller, Nicholas; delToro, Damian; Grimes, Shelley; Jardine, Paul J; Smith, Douglas E

    2014-06-20

    We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine(3+) causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interactions facilitate packaging despite increasing the energy of the theoretical optimum spooled DNA conformation.

  4. Radiobiology with DNA ligands

    International Nuclear Information System (INIS)

    Weinreich, R.; Argentini, M.; Guenther, I.; Koziorowski, J.; Larsson, B.; Nievergelt-Egido, M.C.; Salt, C.; Wyer, L.; Dos Santos, D.F.; Hansen, H.J.

    1997-01-01

    The paper deals with the following topics: labelling of DNA ligands and other tumour-affinic compounds with 4.15-d 124 I, radiotoxicity of Hoechst 33258 and 33342 and of iodinated Hoechst 33258 in cell cultures, preparation of 76 Br-, 123 I-, and 221 At-labelled 5-halo-2'-deoxyuridine, chemical syntheses of boron derivatives of Hoechst 33258.III., Gadolinium neutron capture therapy

  5. DNA nanotechnology and fluorescence applications.

    Science.gov (United States)

    Schlichthaerle, Thomas; Strauss, Maximilian T; Schueder, Florian; Woehrstein, Johannes B; Jungmann, Ralf

    2016-06-01

    Structural DNA nanotechnology allow researchers to use the unique molecular recognition properties of DNA strands to construct nanoscale objects with almost arbitrary complexity in two and three dimensions. Abstracted as molecular breadboards, DNA nanostructures enable nanometer-precise placement of guest molecules such as proteins, fluorophores, or nanoparticles. These assemblies can be used to study biological phenomena with unprecedented control over number, spacing, and molecular identity. Here, we give a general introduction to structural DNA nanotechnology and more specifically discuss applications of DNA nanostructures in the field of fluorescence and plasmonics. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Next generation DNA led technologies

    CERN Document Server

    Jyothsna, G; Kashyap, Amita

    2016-01-01

    This brief highlights advances in DNA technologies and their wider applications. DNA is the source of life and has been studied since a generation, but very little is known as yet. Several sophisticated technologies of the current era have laid their foundations on the principle of DNA based mechanisms. DNA based technologies are bringing a new revolution of Advanced Science and Technology. Forensic Investigation, Medical Diagnosis, Paternity Disputes, Individual Identity, Health insurance, Motor Insurance have incorporated the DNA testing and profiling technologies for settling the issues.

  7. DNA AND ITS METAPHORES

    Directory of Open Access Journals (Sweden)

    Jan Domaradzki

    2015-04-01

    Full Text Available The aim of the present paper is to describe the main metaphors presented in genetic discourse: DNA as text, information, language, book, code, project/blueprint, map, computer, music, and cooking. It also analyses the social implication of these metaphors. The author of this article argues that metaphors are double-edged swords: while they brighten difficult and abstract genetic concepts, they also lead to the misunderstanding and misinterpretation of the reality. The reason for this is that most of these metaphors are of deterministic, reductionist, and fatalistic character. Consequently, they shift the attention from complexity of genetic processes. Moreover, as they appeal to emotions, ascetics, and morality they may involve exaggeration: while they bring hope, they also create an atmosphere of fear over the misuse of genetic knowledge. The author of this article states that the genetic metaphors do not simply reflect the social ideas on DNA, but also shape our understanding of genetics and imagination on the social application of genetic knowledge. Due to this reason, DNA should be understood not only as a biological code, but as a cultural as well.

  8. DNA adducts-chemical addons

    Directory of Open Access Journals (Sweden)

    T R Rajalakshmi

    2015-01-01

    Full Text Available DNA adduct is a piece of DNA covalently bond to a chemical (safrole, benzopyrenediol epoxide, acetaldehyde. This process could be the start of a cancerous cell. When a chemical binds to DNA, it gets damaged resulting in abnormal replication. This could be the start of a mutation and without proper DNA repair, this can lead to cancer. It is this chemical that binds with the DNA is our prime area of concern. Instead of performing the whole body analysis for diagnosing cancer, this test could be carried out for early detection of cancer. When scanning tunneling microscope is used, the DNA results can be obtained earlier. DNA adducts in scientific experiments are used as biomarkers.

  9. Mitochondrial DNA repair and aging

    International Nuclear Information System (INIS)

    Mandavilli, Bhaskar S.; Santos, Janine H.; Van Houten, Bennett

    2002-01-01

    The mitochondrial electron transport chain plays an important role in energy production in aerobic organisms and is also a significant source of reactive oxygen species that damage DNA, RNA and proteins in the cell. Oxidative damage to the mitochondrial DNA is implicated in various degenerative diseases, cancer and aging. The importance of mitochondrial ROS in age-related degenerative diseases is further strengthened by studies using animal models, Caenorhabditis elegans, Drosophila and yeast. Research in the last several years shows that mitochondrial DNA is more susceptible to various carcinogens and ROS when compared to nuclear DNA. DNA damage in mammalian mitochondria is repaired by base excision repair (BER). Studies have shown that mitochondria contain all the enzymes required for BER. Mitochondrial DNA damage, if not repaired, leads to disruption of electron transport chain and production of more ROS. This vicious cycle of ROS production and mtDNA damage ultimately leads to energy depletion in the cell and apoptosis

  10. Mitochondrial DNA repair and aging

    Energy Technology Data Exchange (ETDEWEB)

    Mandavilli, Bhaskar S.; Santos, Janine H.; Van Houten, Bennett

    2002-11-30

    The mitochondrial electron transport chain plays an important role in energy production in aerobic organisms and is also a significant source of reactive oxygen species that damage DNA, RNA and proteins in the cell. Oxidative damage to the mitochondrial DNA is implicated in various degenerative diseases, cancer and aging. The importance of mitochondrial ROS in age-related degenerative diseases is further strengthened by studies using animal models, Caenorhabditis elegans, Drosophila and yeast. Research in the last several years shows that mitochondrial DNA is more susceptible to various carcinogens and ROS when compared to nuclear DNA. DNA damage in mammalian mitochondria is repaired by base excision repair (BER). Studies have shown that mitochondria contain all the enzymes required for BER. Mitochondrial DNA damage, if not repaired, leads to disruption of electron transport chain and production of more ROS. This vicious cycle of ROS production and mtDNA damage ultimately leads to energy depletion in the cell and apoptosis.

  11. Radiation damage of DNA. Model for direct ionization of DNA

    International Nuclear Information System (INIS)

    Kobayashi, Kazuo; Tagawa, Seiichi

    2004-01-01

    Current aspects of radiation damage of DNA, particularly induced by the direct effect of radiation, and author's method of pulse radiolysis are described in relation to behavior of ions formed by radiation and active principles to induce the strand break. In irradiation of DNA solution in water, the direct effect of radiation is derived from ionization of DNA itself and indirect one, from the reaction between DNA and radicals generated from water molecules and the former direct one has been scarcely investigated due to difficulty of experimental approach. Radicals generated in sugar moiety of DNA are shown important in the strand break by recent studies on crystalline DNA irradiated by X-ray, DNA solution by electron and photon beams, hydrated DNA by γ-ray and by high linear energy transfer (LET) ion. Author's pulse radiolysis studies have revealed behaviors of guanine and adenine radical cations in dynamics of DNA oxidation. Since reactions described are the model, the experimental approach is thought necessary for elucidation of the actually occurring DNA damage in living cells. (N.I.)

  12. DNA stable-isotope probing (DNA-SIP).

    Science.gov (United States)

    Dunford, Eric A; Neufeld, Josh D

    2010-08-02

    DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

  13. Dynamics of DNA conformations and DNA-protein interaction

    DEFF Research Database (Denmark)

    Metzler, R.; Ambjörnsson, T.; Lomholt, Michael Andersen

    2005-01-01

    Optical tweezers, atomic force microscopes, patch clamping, or fluorescence techniques make it possible to study both the equilibrium conformations and dynamics of single DNA molecules as well as their interaction with binding proteins. In this paper we address the dynamics of local DNA...... denaturation (bubble breathing), deriving its dynamic response to external physical parameters and the DNA sequence in terms of the bubble relaxation time spectrum and the autocorrelation function of bubble breathing. The interaction with binding proteins that selectively bind to the DNA single strand exposed...... in a denaturation bubble are shown to involve an interesting competition of time scales, varying between kinetic blocking of protein binding up to full binding protein-induced denaturation of the DNA. We will also address the potential to use DNA physics for the design of nanosensors. Finally, we report recent...

  14. DNA adducts as molecular dosimeters

    International Nuclear Information System (INIS)

    Lucier, G.W.

    1990-01-01

    There is compelling evidence that DNA adducts play an important role in the actions of many pulmonary carcinogens. During the last ten years sensitive methods (antibodies and 32 P-postlabeling) have been developed that permit detection of DNA adducts in tissues of animals or humans exposed to low levels of some genotoxic carcinogens. This capability has led to approaches designed to more reliably estimate the shape of the dose-response curve in the low dose region for a few carcinogens. Moreover, dosimetry comparisions can, in some cases, be made between animals and humans which help in judging the adequacy of animal models for human risk assessments. There are several points that need to be considered in the evaluation of DNA adducts as a molecular dosimeter. For example, DNA adduct formation is only one of many events that are needed for tumor development and some potent carcinogens do not form DNA adducts; i.e., TCDD. Other issues that need to be considered are DNA adduct heterogeneity, DNA repair, relationship of DNA adducts to somatic mutation and cell specificity in DNA adduct formation and persistence. Molecular epidemiology studies often require quantitation of adducts in cells such as lymphocytes which may or may not be reliable surrogates for adduct concentrations in target issues. In summary, accurate quantitation of low levels of DNA adducts may provide data useful in species to species extrapolation of risk including the development of more meaningful human monitoring programs

  15. DNA-based watermarks using the DNA-Crypt algorithm

    Science.gov (United States)

    Heider, Dominik; Barnekow, Angelika

    2007-01-01

    Background The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. Results The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. Conclusion The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms. PMID:17535434

  16. Superimposed Code Theorectic Analysis of DNA Codes and DNA Computing

    Science.gov (United States)

    2010-03-01

    that the hybridization that occurs between a DNA strand and its Watson - Crick complement can be used to perform mathematical computation. This research...ssDNA single stranded DNA WC Watson – Crick A Adenine C Cytosine G Guanine T Thymine ... Watson - Crick (WC) duplex, e.g., TCGCA TCGCA . Note that non-WC duplexes can form and such a formation is called a cross-hybridization. Cross

  17. DNA-based watermarks using the DNA-Crypt algorithm

    Directory of Open Access Journals (Sweden)

    Barnekow Angelika

    2007-05-01

    Full Text Available Abstract Background The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. Results The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. Conclusion The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms.

  18. DNA-based watermarks using the DNA-Crypt algorithm.

    Science.gov (United States)

    Heider, Dominik; Barnekow, Angelika

    2007-05-29

    The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms.

  19. The DNA Files

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-06-09

    The DNA Files is a radio documentary which disseminates genetics information over public radio. The documentaries explore subjects which include the following: How genetics affects society. How human life began and how it evolved. Could new prenatal genetic tests hold the key to disease prevention later in life? Would a national genetic data base sacrifice individual privacy? and Should genes that may lead to the cure for cancer be privately owned? This report serves as a project update for the second quarter of 1998. It includes the spring/summer 1998 newsletter, the winter 1998 newsletter, the program clock, and the latest flyer.

  20. Towards a DNA Nanoprocessor: Reusable Tile-Integrated DNA Circuits.

    Science.gov (United States)

    Gerasimova, Yulia V; Kolpashchikov, Dmitry M

    2016-08-22

    Modern electronic microprocessors use semiconductor logic gates organized on a silicon chip to enable efficient inter-gate communication. Here, arrays of communicating DNA logic gates integrated on a single DNA tile were designed and used to process nucleic acid inputs in a reusable format. Our results lay the foundation for the development of a DNA nanoprocessor, a small and biocompatible device capable of performing complex analyses of DNA and RNA inputs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. DNA-Conjugated Organic Chromophores in DNA Stacking Interactions

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V.; Pedersen, Erik Bjerregaard

    2009-01-01

    Since the discovery of the intercalation of acridine derivatives into DNA (1961), chemists have synthesized many intercalators tethered to DNA. Advances in the chemical synthesis of modified nucleosides along with progress in oligonucleotide synthesis have made it possible to introduce organic ch...... review presents those efforts in the design of intercalators/organic chromophores as oligonucleotide conjugates that form a foundation for the generation of novel nucleic acid architectures......Since the discovery of the intercalation of acridine derivatives into DNA (1961), chemists have synthesized many intercalators tethered to DNA. Advances in the chemical synthesis of modified nucleosides along with progress in oligonucleotide synthesis have made it possible to introduce organic...

  2. DNA methylation and memory formation.

    Science.gov (United States)

    Day, Jeremy J; Sweatt, J David

    2010-11-01

    Memory formation and storage require long-lasting changes in memory-related neuronal circuits. Recent evidence indicates that DNA methylation may serve as a contributing mechanism in memory formation and storage. These emerging findings suggest a role for an epigenetic mechanism in learning and long-term memory maintenance and raise apparent conundrums and questions. For example, it is unclear how DNA methylation might be reversed during the formation of a memory, how changes in DNA methylation alter neuronal function to promote memory formation, and how DNA methylation patterns differ between neuronal structures to enable both consolidation and storage of memories. Here we evaluate the existing evidence supporting a role for DNA methylation in memory, discuss how DNA methylation may affect genetic and neuronal function to contribute to behavior, propose several future directions for the emerging subfield of neuroepigenetics, and begin to address some of the broader implications of this work.

  3. Monitoring Biodiversity using Environmental DNA

    DEFF Research Database (Denmark)

    Thomsen, Philip Francis

    DNA). Especially the advance in DNA sequencing technology has revolutionized this field and opened new frontiers in ecology, evolution and environmental sciences. Also, it is becoming a powerful tool for field biologist, with new and efficient methods for monitoring biodiversity. This thesis focuses on the use...... of eDNA in monitoring of biodiversity in different settings. First, it is shown that a diversity of rare freshwater animals – representing amphibians, fish, mammals, insects and crustaceans – can be detected based on eDNA obtained directly from 15 ml water samples of lakes, ponds and streams...... setting, showing that eDNA obtained directly from ½ l seawater samples can account for marine fish biodiversity using NGS. Promisingly, eDNA covered the fish diversity better than any of 9 methods, conventionally used in marine fish surveys. Additionally, it is shown that even short 100-bp. fish e...

  4. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

    Energy Technology Data Exchange (ETDEWEB)

    Haruta, Mayumi [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Shimada, Midori, E-mail: midorism@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Nishiyama, Atsuya; Johmura, Yoshikazu [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Le Tallec, Benoît; Debatisse, Michelle [Institut Curie, Centre de Recherche, 26 rue d’Ulm, CNRS UMR 3244, 75248 ParisCedex 05 (France); Nakanishi, Makoto, E-mail: mkt-naka@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan)

    2016-01-22

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

  5. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

    Science.gov (United States)

    Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L.; Tomkinson, Alan E.; Tainer, John A.; Ellenberger, Tom

    2015-01-01

    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation. PMID:26130724

  6. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

    International Nuclear Information System (INIS)

    Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

    2016-01-01

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

  7. DNA controlled assembly of liposomes

    DEFF Research Database (Denmark)

    Vogel, Stefan; Jakobsen, Ulla; Simonsen, Adam Cohen

    2009-01-01

    DNA-encoding of solid nanoparticles requires surfacechemistry, which is often tedious and not generally applicable. In the present study non-covalently attached DNA are used to assemble soft nanoparticles (liposomes) in solution. This process displays remarkably sharp thermal transitions from...... assembled to disassembled state for which reason this method allows easy and fast detection of polynucleotides (e.g. DNA or RNA), including single nucleotide polymorphisms as well as insertions and deletions....

  8. Biosensors for DNA sequence detection

    Science.gov (United States)

    Vercoutere, Wenonah; Akeson, Mark

    2002-01-01

    DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

  9. DNA damage, homology-directed repair, and DNA methylation.

    Directory of Open Access Journals (Sweden)

    Concetta Cuozzo

    2007-07-01

    Full Text Available To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP genes (DR-GFP. A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

  10. Fluorescence Microscopy of Nanochannel-Confined DNA.

    Science.gov (United States)

    Westerlund, Fredrik; Persson, Fredrik; Fritzsche, Joachim; Beech, Jason P; Tegenfeldt, Jonas O

    2018-01-01

    Stretching of DNA in nanoscale confinement allows for several important studies. The genetic contents of the DNA can be visualized on the single DNA molecule level and both the polymer physics of confined DNA and also DNA/protein and other DNA/DNA-binding molecule interactions can be explored. This chapter describes the basic steps to fabricate the nanostructures, perform the experiments and analyze the data.

  11. Aging and DNA repair capability. [Review

    Energy Technology Data Exchange (ETDEWEB)

    Tice, R R

    1977-01-01

    A review of the literature on DNA repair processes in relation to aging is presented under the following headings: DNA repair processes; age-related occurrence of unrepaired DNA lesions; DNA repair capability as a function of age; tissue-specific DNA repair capability; acceleration of the aging process by exposure to DNA damaging agents; human genetic syndromes; and longevity and DNA repair processes. (HLW)

  12. DNA modification by alkylating compounds

    Energy Technology Data Exchange (ETDEWEB)

    Kruglyakova, E.E.

    1985-09-01

    Results are given for research on the physico-chemical properties of alkylating compounds - nitroso alkyl ureas (NAU) which possess a broad spectrum of biological activity, such as mutagenic, carcinogenic, and anti-tumor action that is due to the alkylation and carbamoylation of DNA as well as other cellular components. Identified chemical products of NAU interaction with DNA and its components are cited. Structural conversions of a DNA macromolecule resulting from its chemical modification are examined. NAU are used to discuss possible biological consequences of DNA modification. 148 references.

  13. Supercoil Formation During DNA Melting

    Science.gov (United States)

    Sayar, Mehmet; Avsaroglu, Baris; Kabakcioglu, Alkan

    2009-03-01

    Supercoil formation plays a key role in determining the structure-function relationship in DNA. Biological and technological processes, such as protein synthesis, polymerase chain reaction, and microarrays relys on separation of the two strands in DNA, which is coupled to the unwinding of the supercoiled structure. This problem has been studied theoretically via Peyrard-Bishop and Poland-Scheraga type models, which include a simple representation of the DNA structural properties. In recent years, computational models, which provide a more realtistic representaion of DNA molecule, have been used to study the melting behavior of short DNA chains. Here, we will present a new coarse-grained model of DNA which is capable of simulating sufficiently long DNA chains for studying the supercoil formation during melting, without sacrificing the local structural properties. Our coarse-grained model successfully reproduces the local geometry of the DNA molecule, such as the 3'-5' directionality, major-minor groove structure, and the helical pitch. We will present our initial results on the dynamics of supercoiling during DNA melting.

  14. Monogenic diseases of DNA repair

    DEFF Research Database (Denmark)

    Keijzers, Guido; Bakula, Daniela; Scheibye-Knudsen, Morten

    2017-01-01

    Maintaining the stability of the genome is essential for all organisms, and it is not surprising that damage to DNA has been proposed as an explanation for multiple chronic diseases.1-5 Conserving a pristine genome is therefore of central importance to our health. To overcome the genotoxic stress...... of a growing number of human diseases. Notably, many of these monogenic DNA-repair disorders display features of accelerated aging, supporting the notion that genome maintenance is a key factor for organismal longevity. This review focuses on the physiological consequences of loss of DNA repair, particularly...... in the context of monogenic DNA-repair diseases....

  15. DNA nanotechnology-enabled biosensors.

    Science.gov (United States)

    Chao, Jie; Zhu, Dan; Zhang, Yinan; Wang, Lianhui; Fan, Chunhai

    2016-02-15

    Biosensors employ biological molecules to recognize the target and utilize output elements which can translate the biorecognition event into electrical, optical or mass-sensitive signals to determine the quantities of the target. DNA-based biosensors, as a sub-field to biosensor, utilize DNA strands with short oligonucleotides as probes for target recognition. Although DNA-based biosensors have offered a promising alternative for fast, simple and cheap detection of target molecules, there still exist key challenges including poor stability and reproducibility that hinder their competition with the current gold standard for DNA assays. By exploiting the self-recognition properties of DNA molecules, researchers have dedicated to make versatile DNA nanostructures in a highly rigid, controllable and functionalized manner, which offers unprecedented opportunities for developing DNA-based biosensors. In this review, we will briefly introduce the recent advances on design and fabrication of static and dynamic DNA nanostructures, and summarize their applications for fabrication and functionalization of DNA-based biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Statistical Approaches for DNA Barcoding

    DEFF Research Database (Denmark)

    Nielsen, Rasmus; Matz, M.

    2006-01-01

    The use of DNA as a tool for species identification has become known as "DNA barcoding" (Floyd et al., 2002; Hebert et al., 2003; Remigio and Hebert, 2003). The basic idea is straightforward: a small amount of DNA is extracted from the specimen, amplified and sequenced. The gene region sequenced...... is chosen so that it is nearly identical among individuals of the same species, but different between species, and therefore its sequence, can serve as an identification tag for the species ("DNA barcode"). By matching the sequence obtained from an unidentified specimen ("query" sequence) to the database...

  17. Radiobiological significance of DNA repair

    International Nuclear Information System (INIS)

    Kuzin, A.M.

    1978-01-01

    A short outline is given on the history of the problem relating to the repair of radiation injuries, specifically its molecular mechanisms. The most urgent problems which currently confront the researchers are noted. This is a further study on the role of DNA repair in post-radiation recovery, search for ways to activate and suppress DNA repair, investigations into the activity balance of various repair enzymes as well as the problem of errors in the structure of repairing DNA. An important role is attached to the investigations of DNA repair in solving a number of practical problems

  18. DNA repair: keeping it together

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2004-01-01

    A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest.......A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest....

  19. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication.

    Science.gov (United States)

    Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

    2016-01-22

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Clinical strains of acinetobacter classified by DNA-DNA hybridization

    International Nuclear Information System (INIS)

    Tjernberg, I.; Ursing, J.

    1989-01-01

    A collection of Acinetobacter strains consisting of 168 consecutive clinical strains and 30 type and reference strains was studied by DNA-DNA hybridization and a few phenotypic tests. The field strains could be allotted to 13 DNA groups. By means of reference strains ten of these could be identified with groups described by Bouvet and Grimont (1986), while three groups were new; they were given the numbers 13-15. The type strain of A. radioresistens- recently described by Nishimura et al. (1988) - was shown to be a member of DNA group 12, which comprised 31 clinical isolates. Of the 19 strains of A. junii, eight showed hemolytic acitivity on sheep and human blood agar and an additional four strains on human blood agar only. Strains of this species have previously been regarded as non-hemolytic. Reciprocal DNA pairing data for the reference strains of the DNA gropus were treated by UPGMA clustering. The reference strains for A. calcoaceticus, A. baumannii and DNA groups 3 and 13 formed a cluster with about 70% relatedness within the cluster. Other DNA groups joined at levels below 60%. (author)

  1. Clinical strains of acinetobacter classified by DNA-DNA hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Tjernberg, I; Ursing, J [Department of Medical Microbiology, University of Lund, Malmoe General Hospital, Malmoe (Sweden)

    1989-01-01

    A collection of Acinetobacter strains consisting of 168 consecutive clinical strains and 30 type and reference strains was studied by DNA-DNA hybridization and a few phenotypic tests. The field strains could be allotted to 13 DNA groups. By means of reference strains ten of these could be identified with groups described by Bouvet and Grimont (1986), while three groups were new; they were given the numbers 13-15. The type strain of A. radioresistens- recently described by Nishimura et al. (1988) - was shown to be a member of DNA group 12, which comprised 31 clinical isolates. Of the 19 strains of A. junii, eight showed hemolytic acitivity on sheep and human blood agar and an additional four strains on human blood agar only. Strains of this species have previously been regarded as non-hemolytic. Reciprocal DNA pairing data for the reference strains of the DNA gropus were treated by UPGMA clustering. The reference strains for A. calcoaceticus, A. baumannii and DNA groups 3 and 13 formed a cluster with about 70% relatedness within the cluster. Other DNA groups joined at levels below 60%. (author).

  2. Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage phi29

    OpenAIRE

    Keller, Nicholas; delToro, Damian; Grimes, Shelley; Jardine, Paul J.; Smith, Douglas E.

    2014-01-01

    We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine3+ causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interacti...

  3. Master equation approach to DNA breathing in heteropolymer DNA

    DEFF Research Database (Denmark)

    Ambjörnsson, Tobias; Banik, Suman K; Lomholt, Michael A

    2007-01-01

    After crossing an initial barrier to break the first base-pair (bp) in double-stranded DNA, the disruption of further bps is characterized by free energies up to a few k(B)T. Thermal motion within the DNA double strand therefore causes the opening of intermittent single-stranded denaturation zones......, the DNA bubbles. The unzipping and zipping dynamics of bps at the two zipper forks of a bubble, where the single strand of the denatured zone joins the still intact double strand, can be monitored by single molecule fluorescence or NMR methods. We here establish a dynamic description of this DNA breathing...... in a heteropolymer DNA with given sequence in terms of a master equation that governs the time evolution of the joint probability distribution for the bubble size and position along the sequence. The transfer coefficients are based on the Poland-Scheraga free energy model. We derive the autocorrelation function...

  4. DNA damage in neurodegenerative diseases

    Energy Technology Data Exchange (ETDEWEB)

    Coppedè, Fabio, E-mail: fabio.coppede@med.unipi.it; Migliore, Lucia, E-mail: lucia.migliore@med.unipi.it

    2015-06-15

    Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

  5. Authentication of forensic DNA samples.

    Science.gov (United States)

    Frumkin, Dan; Wasserstrom, Adam; Davidson, Ariane; Grafit, Arnon

    2010-02-01

    Over the past twenty years, DNA analysis has revolutionized forensic science, and has become a dominant tool in law enforcement. Today, DNA evidence is key to the conviction or exoneration of suspects of various types of crime, from theft to rape and murder. However, the disturbing possibility that DNA evidence can be faked has been overlooked. It turns out that standard molecular biology techniques such as PCR, molecular cloning, and recently developed whole genome amplification (WGA), enable anyone with basic equipment and know-how to produce practically unlimited amounts of in vitro synthesized (artificial) DNA with any desired genetic profile. This artificial DNA can then be applied to surfaces of objects or incorporated into genuine human tissues and planted in crime scenes. Here we show that the current forensic procedure fails to distinguish between such samples of blood, saliva, and touched surfaces with artificial DNA, and corresponding samples with in vivo generated (natural) DNA. Furthermore, genotyping of both artificial and natural samples with Profiler Plus((R)) yielded full profiles with no anomalies. In order to effectively deal with this problem, we developed an authentication assay, which distinguishes between natural and artificial DNA based on methylation analysis of a set of genomic loci: in natural DNA, some loci are methylated and others are unmethylated, while in artificial DNA all loci are unmethylated. The assay was tested on natural and artificial samples of blood, saliva, and touched surfaces, with complete success. Adopting an authentication assay for casework samples as part of the forensic procedure is necessary for maintaining the high credibility of DNA evidence in the judiciary system.

  6. Effects of radiation on DNA

    International Nuclear Information System (INIS)

    Medina, V.F.O.

    1978-01-01

    Irradiation has been shown to depress DNA (deoxyribonucleic acid) synthesis resulting in deficient DNA synthesis. In one experiment, Hela S 3 cells completed the next division after a dose of 500 rads to 200 kw X-rays. Another experiment showed that the amount of DNA synthesized was dependent on the stage in the generation cycle at which the cells are irradiated (Giffites and Tolmach, 1975). DNA synthesis was measured by radioactive thymidine incorporation. The smallest deficiency (20-35%) after a dose of 500 rad X-ray was observed in Hela S 3 cells irradiated in early G 1 or early G 2 , while the greatest deficiency (55-70*) after 500 rad X-ray was found in cells irradiated at mitosis or at the Gsub(1)/S transition. Using velocity sedimentation in alkaline gradients of the DNA from hamster, Elkind, et al 1972, studied repair processes as a function of X-ray dose. DNA containing material released by alkaline lysis was found initially contained in a complex-containing lipid, the sedimentation of which was anomalous relative to denatured RNA from unirradated cells. Doses of X-rays small enough to be in the range that permits high survival (100-800 rads) speed the resolution of single-stranded DNA from this DNA complex, giving rise to a species having a number average molecular weight of 2 x 10 8 daltons. Larger doses greater than 1000 to 2000 rads resulted in a degradation of these DNA strands. Incubation after irradiation resulted in the rapid repair of damage, although the rate of repair of damage to the complex resulted in a reassociation of lipid and DNA. This evidence supports the possibility that a large DNA-membrane structure is a principal target of radiation

  7. DNA damage in neurodegenerative diseases

    International Nuclear Information System (INIS)

    Coppedè, Fabio; Migliore, Lucia

    2015-01-01

    Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

  8. DNA Movies and Panspermia

    Directory of Open Access Journals (Sweden)

    Victor Norris

    2011-10-01

    Full Text Available There are several ways that our species might try to send a message to another species separated from us by space and/or time. Synthetic biology might be used to write an epitaph to our species, or simply “Kilroy was here”, in the genome of a bacterium via the patterns of either (1 the codons to exploit Life's non-equilibrium character or (2 the bases themselves to exploit Life's quasi-equilibrium character. We suggest here how DNA movies might be designed using such patterns. We also suggest that a search for mechanisms to create and preserve such patterns might lead to a better understanding of modern cells. Finally, we argue that the cutting-edge microbiology and synthetic biology needed for the Kilroy project would put origin-of-life studies in the vanguard of research.

  9. DNA: Polymer and molecular code

    Science.gov (United States)

    Shivashankar, G. V.

    1999-10-01

    The thesis work focusses upon two aspects of DNA, the polymer and the molecular code. Our approach was to bring single molecule micromanipulation methods to the study of DNA. It included a home built optical microscope combined with an atomic force microscope and an optical tweezer. This combined approach led to a novel method to graft a single DNA molecule onto a force cantilever using the optical tweezer and local heating. With this method, a force versus extension assay of double stranded DNA was realized. The resolution was about 10 picoN. To improve on this force measurement resolution, a simple light backscattering technique was developed and used to probe the DNA polymer flexibility and its fluctuations. It combined the optical tweezer to trap a DNA tethered bead and the laser backscattering to detect the beads Brownian fluctuations. With this technique the resolution was about 0.1 picoN with a millisecond access time, and the whole entropic part of the DNA force-extension was measured. With this experimental strategy, we measured the polymerization of the protein RecA on an isolated double stranded DNA. We observed the progressive decoration of RecA on the l DNA molecule, which results in the extension of l , due to unwinding of the double helix. The dynamics of polymerization, the resulting change in the DNA entropic elasticity and the role of ATP hydrolysis were the main parts of the study. A simple model for RecA assembly on DNA was proposed. This work presents a first step in the study of genetic recombination. Recently we have started a study of equilibrium binding which utilizes fluorescence polarization methods to probe the polymerization of RecA on single stranded DNA. In addition to the study of material properties of DNA and DNA-RecA, we have developed experiments for which the code of the DNA is central. We studied one aspect of DNA as a molecular code, using different techniques. In particular the programmatic use of template specificity makes

  10. DNA Extraction Techniques for Use in Education

    Science.gov (United States)

    Hearn, R. P.; Arblaster, K. E.

    2010-01-01

    DNA extraction provides a hands-on introduction to DNA and enables students to gain real life experience and practical knowledge of DNA. Students gain a sense of ownership and are more enthusiastic when they use their own DNA. A cost effective, simple protocol for DNA extraction and visualization was devised. Buccal mucosal epithelia provide a…

  11. Forensic trace DNA: A review

    NARCIS (Netherlands)

    R.A.H. van Oorschot (Roland ); K. Ballantyne (Kaye); R.J. Mitchell (R. John)

    2010-01-01

    textabstractDNA analysis is frequently used to acquire information from biological material to aid enquiries associated with criminal offences, disaster victim identification and missing persons investigations. As the relevance and value of DNA profiling to forensic investigations has increased, so

  12. Aktionslæringens DNA

    DEFF Research Database (Denmark)

    Madsen, Benedicte

    Aktionslæringen DNA giver en række redskaber til læring i fællesskaber, uanset om der arbejdes med individuelle eller kollektive projekter i offentlig eller privat regi. Metoden danner modvægt til de mere individuelistiske traditioner inden for voksenpædagogikken. DNA-metaforen bruges bogen igennem...

  13. LEGO-like DNA Structures

    DEFF Research Database (Denmark)

    Gothelf, Kurt Vesterager

    2012-01-01

    -dimensional (3D) DNA structures by self-assembly of single-stranded DNA “bricks.” The method opens a new route to complex self-assembled (3D) nanostructures that may serve as addressable templates for placing guest molecules with high precision, with possible applications in biophysics, medicine...

  14. Replication of bacteriophage lambda DNA

    International Nuclear Information System (INIS)

    Tsurimoto, T.; Matsubara, K.

    1983-01-01

    In this paper results of studies on the mechanism of bacteriophage lambda replication using molecular biological and biochemical approaches are reported. The purification of the initiator proteins, O and P, and the role of the O and P proteins in the initiation of lambda DNA replication through interactions with specific DNA sequences are described. 47 references, 15 figures

  15. DNA adducts in senescent cells

    International Nuclear Information System (INIS)

    Gaubatz, J.W.

    1987-01-01

    Perturbations in DNA repair and other metabolic processes during development and aging might affect the steady-state level of genomic damage. The persistence or accumulation of DNA lesions in postmitotic cells could have a significant impact on proper cellular function, interfering with gene regulation for example. To test the notion that DNA damage increases as a function of age in non-dividing cells, DNA was purified from heart tissue of C57BL/6Nia mice at different ages and analyzed by post labeling techniques to detect DNA adducts. In the present experiments, four-dimensional, thin-layer chromatography was used to isolate aromatic adducts that were labeled with carrier-free (γ- 32 P) ATP under DNA-P excess conditions. The complexity and frequency of aromatic adducts varied between DNA samples. Several adducts were present in all preparations and were clearly more abundant in nucleotide maps of mature and old heart DNA. However, a direct correlation with age was not observed. In contrast, experiments in which aromatic adducts were first isolated by phase-transfer to 1-butanol, then labeled with excess (γ- 32 P)ATP indicated that there was an age-related increase in these adducts. The results are consistent with their earlier studies that showed alkyl adducts increased during aging of mouse myocardium and suggest that a common repair pathway might be involved

  16. Authenticity in ancient DNA studies

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Willerslev, Eske

    2006-01-01

    Ancient DNA studies represent a powerful tool that can be used to obtain genetic insights into the past. However, despite the publication of large numbers of apparently successful ancient DNA studies, a number of problems exist with the field that are often ignored. Therefore, questions exist as ...

  17. Bubble coalescence in breathing DNA

    DEFF Research Database (Denmark)

    Novotný, Tomas; Pedersen, Jonas Nyvold; Ambjörnsson, Tobias

    2007-01-01

    We investigate the coalescence of two DNA bubbles initially located at weak segments and separated by a more stable barrier region in a designed construct of double-stranded DNA. The characteristic time for bubble coalescence and the corresponding distribution are derived, as well as the distribu...... vicious walkers in opposite potentials....

  18. DNA nanotechnology: a future perspective

    Science.gov (United States)

    2013-01-01

    In addition to its genetic function, DNA is one of the most distinct and smart self-assembling nanomaterials. DNA nanotechnology exploits the predictable self-assembly of DNA oligonucleotides to design and assemble innovative and highly discrete nanostructures. Highly ordered DNA motifs are capable of providing an ultra-fine framework for the next generation of nanofabrications. The majority of these applications are based upon the complementarity of DNA base pairing: adenine with thymine, and guanine with cytosine. DNA provides an intelligent route for the creation of nanoarchitectures with programmable and predictable patterns. DNA strands twist along one helix for a number of bases before switching to the other helix by passing through a crossover junction. The association of two crossovers keeps the helices parallel and holds them tightly together, allowing the assembly of bigger structures. Because of the DNA molecule's unique and novel characteristics, it can easily be applied in a vast variety of multidisciplinary research areas like biomedicine, computer science, nano/optoelectronics, and bionanotechnology. PMID:23497147

  19. Multiscale modelling of DNA mechanics

    Czech Academy of Sciences Publication Activity Database

    Dršata, Tomáš; Lankaš, Filip

    2015-01-01

    Roč. 27, č. 32 (2015), 323102/1-323102/12 ISSN 0953-8984 R&D Projects: GA ČR(CZ) GA14-21893S Institutional support: RVO:61388963 Keywords : DNA elasticity * DNA coarse-grained models * molecular dynamics simulations Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.209, year: 2015

  20. DNA damage by Auger emitters

    International Nuclear Information System (INIS)

    Martin, R.F.; d'Cunha, Glenn; Gibbs, Richard; Murray, Vincent; Pardee, Marshall; Allen, B.J.

    1988-01-01

    125 I atoms can be introduced at specific locations along a defined DNA target molecule, either by site-directed incorporation of an 125 I-labelled deoxynucleotide or by binding of an 125 I-labelled sequence-selective DNA ligand. After allowing accumulation of 125 I decay-induced damage to the DNA, application of DNA sequencing techniques enables positions of strand breaks to be located relative to the site of decay, at a resolution corresponding to the distance between adjacent nucleotides [0.34 nm]. Thus, DNA provides a molecular framework to analyse the extent of damage following [averaged] individual decay events. Results can be compared with energy deposition data generated by computer-simulation methods developed by Charlton et al. The DNA sequencing technique also provides information about the chemical nature of the termini of the DNA chains produced following Auger decay-induced damage. In addition to reviewing the application of this approach to the analysis of 125 I decay induced DNA damage, some more recent results obtained by using 67 Ga are also presented. (author)

  1. DNA-extractie zonder remming

    NARCIS (Netherlands)

    Bonants, P.J.M.; Lee, van der T.A.J.

    2011-01-01

    Moleculaire technieken voor de detectie en identificatie van plantenpathogenen maken gebruik van het DNA of RNA van de ziekteverwekker. Voor een aantal substraten, zoals grond, is de extractie van amplificeerbaar nucleïnezuur een probleem. Tijdens de DNA-extractie uit sommige moeilijke substraten

  2. The journey of DNA repair.

    Science.gov (United States)

    Saini, Natalie

    2015-12-01

    21 years ago, the DNA Repair Enzyme was declared "Molecule of the Year". Today, we are celebrating another "year of repair", with the 2015 Nobel Prize in Chemistry being awarded to Aziz Sancar, Tomas Lindahl and Paul Modrich for their collective work on the different DNA repair pathways.

  3. The DnaA Tale

    DEFF Research Database (Denmark)

    Hansen, Flemming G.; Atlung, Tove

    2018-01-01

    , and translation efficiency, as well as, the DnaA protein, its concentration, its binding to DnaA-boxes, and its binding of ATP or ADP. Furthermore, we will discuss the different models for regulation of initiation which have been proposed over the years, with particular emphasis on the Initiator Titration Model....

  4. Effects of radiation on DNA

    International Nuclear Information System (INIS)

    Braddock, M.

    1985-07-01

    The hydroxyl radical (OH radical) is the most damaging radical produced by the effect of ionizing radiation in water. The rate of reaction of the OH radical with purified, native and isodisperse DNA has been determined as compared with calf thymus DNA. This has been achieved by direct observation of the rate of formation of the DNA-OH radical adduct, and by competition with SCN - . Results obtained from direct observation are consistent with calculations which have been performed using the encounter frequency model of Braams and Ebert. However, results obtained for OH radical with DNA derived from competition plots suggest a rate constant somewhat lower than that obtained from direct observation. The relative merits of both techniques are discussed. In order to study the effect of energy deposited directly in the DNA, dry films of purified plasmid DNA have been irradiated in a system where the indirect effects of radical interaction have been minimized. The present results indicate that with different molecular lengths of plasmid DNA, non-random breakage may occur, and that additional damage may be brought about at sites of previously existing damage. Differences in the sensitivity of plasmid DNA molecules of varying lengths to radiation induced double strand breaks have been demonstrated. (author)

  5. The journey of DNA repair

    OpenAIRE

    Saini, Natalie

    2015-01-01

    21 years ago, the DNA Repair Enzyme was declared “Molecule of the Year”. Today, we are celebrating another “year of repair”, with the 2015 Nobel Prize in Chemistry being awarded to Aziz Sancar, Tomas Lindahl and Paul Modrich for their collective work on the different DNA repair pathways.

  6. Drug Addiction and DNA Modifications.

    Science.gov (United States)

    Brown, Amber N; Feng, Jian

    2017-01-01

    Drug addiction is a complex disorder which can be influenced by both genetic and environmental factors. Research has shown that epigenetic modifications can translate environmental signals into changes in gene expression, suggesting that epigenetic changes may underlie the causes and possibly treatment of substance use disorders. This chapter will focus on epigenetic modifications to DNA, which include DNA methylation and several recently defined additional DNA epigenetic changes. We will discuss the functions of DNA modifications and methods for detecting them, followed by a description of the research investigating the function and consequences of drug-induced changes in DNA methylation patterns. Understanding these epigenetic changes may provide us translational tools for the diagnosis and treatment of addiction in the future.

  7. DNA methylation in metabolic disorders

    DEFF Research Database (Denmark)

    Barres, Romain; Zierath, Juleen R

    2011-01-01

    DNA methylation is a major epigenetic modification that controls gene expression in physiologic and pathologic states. Metabolic diseases such as diabetes and obesity are associated with profound alterations in gene expression that are caused by genetic and environmental factors. Recent reports...... have provided evidence that environmental factors at all ages could modify DNA methylation in somatic tissues, which suggests that DNA methylation is a more dynamic process than previously appreciated. Because of the importance of lifestyle factors in metabolic disorders, DNA methylation provides...... a mechanism by which environmental factors, including diet and exercise, can modify genetic predisposition to disease. This article considers the current evidence that defines a role for DNA methylation in metabolic disorders....

  8. Ancient DNA from marine mammals

    DEFF Research Database (Denmark)

    Foote, Andrew David; Hofreiter, Michael; Morin, Philip A.

    2012-01-01

    such as bone, tooth, baleen, skin, fur, whiskers and scrimshaw using ancient DNA (aDNA) approaches provide an oppor- tunity for investigating such changes over evolutionary and ecological timescales. Here, we review the application of aDNA techniques to the study of marine mammals. Most of the studies have...... focused on detecting changes in genetic diversity following periods of exploitation and environmental change. To date, these studies have shown that even small sample sizes can provide useful information on historical genetic diversity. Ancient DNA has also been used in investigations of changes...... in distribution and range of marine mammal species; we review these studies and discuss the limitations of such ‘presence only’ studies. Combining aDNA data with stable isotopes can provide further insights into changes in ecology and we review past studies and suggest future potential applications. We also...

  9. DNA typing by capillary electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, N.

    1997-10-08

    Capillary electrophoresis is becoming more and more important in nucleic acid analysis including DNA sequencing, typing and disease gene measurements. This work summarized the background of DNA typing. The recent development of capillary electrophoresis was also discussed. The second part of the thesis showed the principle of DNA typing based on using the allelic ladder as the absolute standard ladder in capillary electrophoresis system. Future work will be focused on demonstrating DNA typing on multiplex loci and examples of disease diagnosis in the on-line format of PCR-CE. Also capillary array electrophoresis system should allow high throughput, fast speed DNA typing. Only the introduction and conclusions for this report are available here. A reprint was removed for separate processing.

  10. Multiscale modelling of DNA mechanics

    International Nuclear Information System (INIS)

    Dršata, Tomáš; Lankaš, Filip

    2015-01-01

    Mechanical properties of DNA are important not only in a wide range of biological processes but also in the emerging field of DNA nanotechnology. We review some of the recent developments in modeling these properties, emphasizing the multiscale nature of the problem. Modern atomic resolution, explicit solvent molecular dynamics simulations have contributed to our understanding of DNA fine structure and conformational polymorphism. These simulations may serve as data sources to parameterize rigid base models which themselves have undergone major development. A consistent buildup of larger entities involving multiple rigid bases enables us to describe DNA at more global scales. Free energy methods to impose large strains on DNA, as well as bead models and other approaches, are also briefly discussed. (topical review)

  11. Molecular mechanisms of DNA photodamage

    Energy Technology Data Exchange (ETDEWEB)

    Starrs, S.M

    2000-05-01

    Photodamage in DNA, caused by ultraviolet (UV) light, can occur by direct excitation of the nucleobases or indirectly via the action of photosensitisers. Such, DNA photodamage can be potentially mutagenic or lethal. Among the methods available for detecting UV-induced DNA damage, gel sequencing protocols, utilising synthetic oligodeoxyribonucleotides as targets for UV radiation, allow photolesions to be mapped at nucleotide resolution. This approach has been applied to investigate both DNA damage mechanisms. Following a general overview of DNA photoreactivity, and a description of the main experimental procedures, Chapter 3 identifies the origin of an anomalous mobility shift observed in purine chemical sequence ladders that can confuse the interpretation of DNA cleavage results; measures to abolish this shift are also described. Chapters 4 and 5 examine the alkali-labile DNA damage photosensitised by representative nonsteroidal antiinflammatory drugs (NSAIDs) and the fluoroquinolone antibiotics. Suprofen was the most photoactive NSAID studied, producing different patterns of guanine-specific damage in single-stranded and duplex DNA. Uniform modification of guanine bases, typifying attack by singlet oxygen, was observed in single-stranded oligodeoxyribonucleotides. In duplex molecules, modification was limited to the 5'-G of GG doublets, which is indicative of an electron transfer. The effect of quenchers and photoproduct analysis substantiated these findings. The quinolone, nalidixic acid, behaves similarly. The random base cleavage photosensitised by the fluoroquinolones, has been attributed to free radicals produced during their photodecomposition. Chapter 6 addresses the photoreactivity of purines within unusual DNA structures formed by the repeat sequences (GGA){sub n} and (GA){sub n}, and a minihairpin. There was no definitive evidence for enhanced purine reactivity caused by direct excitation. Finally, Chapter 7 investigates the mutagenic potential of a

  12. Molecular mechanisms of DNA photodamage

    International Nuclear Information System (INIS)

    Starrs, S.M.

    2000-05-01

    Photodamage in DNA, caused by ultraviolet (UV) light, can occur by direct excitation of the nucleobases or indirectly via the action of photosensitisers. Such, DNA photodamage can be potentially mutagenic or lethal. Among the methods available for detecting UV-induced DNA damage, gel sequencing protocols, utilising synthetic oligodeoxyribonucleotides as targets for UV radiation, allow photolesions to be mapped at nucleotide resolution. This approach has been applied to investigate both DNA damage mechanisms. Following a general overview of DNA photoreactivity, and a description of the main experimental procedures, Chapter 3 identifies the origin of an anomalous mobility shift observed in purine chemical sequence ladders that can confuse the interpretation of DNA cleavage results; measures to abolish this shift are also described. Chapters 4 and 5 examine the alkali-labile DNA damage photosensitised by representative nonsteroidal antiinflammatory drugs (NSAIDs) and the fluoroquinolone antibiotics. Suprofen was the most photoactive NSAID studied, producing different patterns of guanine-specific damage in single-stranded and duplex DNA. Uniform modification of guanine bases, typifying attack by singlet oxygen, was observed in single-stranded oligodeoxyribonucleotides. In duplex molecules, modification was limited to the 5'-G of GG doublets, which is indicative of an electron transfer. The effect of quenchers and photoproduct analysis substantiated these findings. The quinolone, nalidixic acid, behaves similarly. The random base cleavage photosensitised by the fluoroquinolones, has been attributed to free radicals produced during their photodecomposition. Chapter 6 addresses the photoreactivity of purines within unusual DNA structures formed by the repeat sequences (GGA) n and (GA) n , and a minihairpin. There was no definitive evidence for enhanced purine reactivity caused by direct excitation. Finally, Chapter 7 investigates the mutagenic potential of a dimeric

  13. RAD51 interconnects between DNA replication, DNA repair and immunity.

    Science.gov (United States)

    Bhattacharya, Souparno; Srinivasan, Kalayarasan; Abdisalaam, Salim; Su, Fengtao; Raj, Prithvi; Dozmorov, Igor; Mishra, Ritu; Wakeland, Edward K; Ghose, Subroto; Mukherjee, Shibani; Asaithamby, Aroumougame

    2017-05-05

    RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. DNA Topology and the Initiation of Virus DNA Packaging.

    Directory of Open Access Journals (Sweden)

    Choon Seok Oh

    Full Text Available During progeny assembly, viruses selectively package virion genomes from a nucleic acid pool that includes host nucleic acids. For large dsDNA viruses, including tailed bacteriophages and herpesviruses, immature viral DNA is recognized and translocated into a preformed icosahedral shell, the prohead. Recognition involves specific interactions between the viral packaging enzyme, terminase, and viral DNA recognition sites. Generally, viral DNA is recognized by terminase's small subunit (TerS. The large terminase subunit (TerL contains translocation ATPase and endonuclease domains. In phage lambda, TerS binds a sequence repeated three times in cosB, the recognition site. TerS binding to cosB positions TerL to cut the concatemeric DNA at the adjacent nicking site, cosN. TerL introduces staggered nicks in cosN, generating twelve bp cohesive ends. Terminase separates the cohesive ends and remains bound to the cosB-containing end, in a nucleoprotein structure called Complex I. Complex I docks on the prohead's portal vertex and translocation ensues. DNA topology plays a role in the TerSλ-cosBλ interaction. Here we show that a site, I2, located between cosN and cosB, is critically important for an early DNA packaging step. I2 contains a complex static bend. I2 mutations block DNA packaging. I2 mutant DNA is cut by terminase at cosN in vitro, but in vivo, no cos cleavage is detected, nor is there evidence for Complex I. Models for what packaging step might be blocked by I2 mutations are presented.

  15. The role of DNA dependent protein kinase in synapsis of DNA ends

    NARCIS (Netherlands)

    E.P.W.C. Weterings (Eric); N.S. Verkaik (Nicole); H.T. Brüggenwirth (Hennie); D.C. van Gent (Dik); J.H.J. Hoeijmakers (Jan)

    2003-01-01

    textabstractDNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks

  16. Human mitochondrial DNA (mtDNA) types in Malaysia

    International Nuclear Information System (INIS)

    Lian, L.H.; Koh, C.L.; Lim, M.E.

    2000-01-01

    Each human cell contains hundreds of mitochondria and thousands of double-stranded circular mtDNA. The delineation of human mtDNA variation and genetics over the past decade has provided unique and often startling insights into human evolution, degenerative diseases, and aging. Each mtDNA of 16,569 base pairs, encodes 13 polypeptides essential to the enzymes of the mitochondrial energy generating pathway, plus the necessary tRNAs and rRNAs. The highly polymorphic noncoding D-(displacement) loop region, also called the control region, is approximately 1.2 kb long. It contains two well-characterized hypervariable (HV-) regions, HV1 and HV2. MtDNA identification is usually based on these sequence differences. According to the TWTGDAM (Technical Working Group for DNA Analysis Methods), the minimum requirement for a mtDNA database for HV1 is from positions 16024 to 16365 and for HV2, from positions 00073 to 00340. The targeted Malaysian population subgroups for this study were mainly the Malays, Chinese, Indians, and indigenous Ibans, Bidayuhs, Kadazan-Dusuns, and Bajaus. Research methodologies undertaken included DNA extraction of samples from unrelated individuals, amplification of the specific regions via the polymerase chain reaction (PCR), and preparation of template DNA for sequencing by using an automated DNA sequencer. Sufficient nucleotide sequence data were generated from the mtDNA analysis. When the sequences were analyzed, sequence variations were found to be caused by nucleotide substitutions, insertions, and deletions. Of the three causes of the sequence variations, nucleotide substitutions (86.1%) accounted for the vast majority of polymorphism. It is noted that transitions (83.5%) were predominant when compared to the significantly lower frequencies of transversions (2.6%). Insertions (0.9%) and deletions (13.0%) were rather rare and found only in HV2. The data generated will also form the basis of a Malaysian DNA sequence database of mtDNA D

  17. DNA Replication Profiling Using Deep Sequencing.

    Science.gov (United States)

    Saayman, Xanita; Ramos-Pérez, Cristina; Brown, Grant W

    2018-01-01

    Profiling of DNA replication during progression through S phase allows a quantitative snap-shot of replication origin usage and DNA replication fork progression. We present a method for using deep sequencing data to profile DNA replication in S. cerevisiae.

  18. Programme DNA Lattices: Design, Synthesis and Applications

    National Research Council Canada - National Science Library

    Reif, John

    2006-01-01

    .... Self-assembled DNA nanostructures provide a methodology for bottom-up nanoscale construction of highly patterned systems, utilizing macromolecular DNA tiles" composed of branched DNA, self-assembled...

  19. Single gene retrieval from thermally degraded DNA

    Indian Academy of Sciences (India)

    Unknown

    DNA thermal degradation was shown to occur via a singlet oxygen pathway. A comparative study of the ther- mal degradation of cellular DNA and isolated DNA showed that cellular ..... definite level of energy (e.g. depurination active energy,.

  20. Molecular biological mechanisms I. DNA repair

    International Nuclear Information System (INIS)

    Friedl, A.A.

    2000-01-01

    Cells of all living systems possess a variety of mechanisms that allow to repair spontaneous and exogeneously induced DNA damage. DNA repair deficiencies may invoke enhanced sensitivity towards DNA-damaging agents such as ionizing radiation. They may also enhance the risk of cancer development, both spontaneously or after induction. This article reviews several DNA repair mechanisms, especially those dealing with DNA double-strand breaks, and describes hereditary diseases associated with DNA repair defects. (orig.) [de

  1. DNA Binding by the Ribosomal DNA Transcription Factor Rrn3 Is Essential for Ribosomal DNA Transcription*

    Science.gov (United States)

    Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H.; Rothblum, Katrina; Schneider, David A.; Rothblum, Lawrence I.

    2013-01-01

    The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382–400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I. PMID:23393135

  2. DNA binding by the ribosomal DNA transcription factor rrn3 is essential for ribosomal DNA transcription.

    Science.gov (United States)

    Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H; Rothblum, Katrina; Schneider, David A; Rothblum, Lawrence I

    2013-03-29

    The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382-400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I.

  3. DNA Damage, Mutagenesis and Cancer

    Directory of Open Access Journals (Sweden)

    Ashis K. Basu

    2018-03-01

    Full Text Available A large number of chemicals and several physical agents, such as UV light and γ-radiation, have been associated with the etiology of human cancer. Generation of DNA damage (also known as DNA adducts or lesions induced by these agents is an important first step in the process of carcinogenesis. Evolutionary processes gave rise to DNA repair tools that are efficient in repairing damaged DNA; yet replication of damaged DNA may take place prior to repair, particularly when they are induced at a high frequency. Damaged DNA replication may lead to gene mutations, which in turn may give rise to altered proteins. Mutations in an oncogene, a tumor-suppressor gene, or a gene that controls the cell cycle can generate a clonal cell population with a distinct advantage in proliferation. Many such events, broadly divided into the stages of initiation, promotion, and progression, which may occur over a long period of time and transpire in the context of chronic exposure to carcinogens, can lead to the induction of human cancer. This is exemplified in the long-term use of tobacco being responsible for an increased risk of lung cancer. This mini-review attempts to summarize this wide area that centers on DNA damage as it relates to the development of human cancer.

  4. Minisequencing mitochondrial DNA pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Carracedo Ángel

    2008-04-01

    Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

  5. Ancient and modern environmental DNA

    Science.gov (United States)

    Pedersen, Mikkel Winther; Overballe-Petersen, Søren; Ermini, Luca; Sarkissian, Clio Der; Haile, James; Hellstrom, Micaela; Spens, Johan; Thomsen, Philip Francis; Bohmann, Kristine; Cappellini, Enrico; Schnell, Ida Bærholm; Wales, Nathan A.; Carøe, Christian; Campos, Paula F.; Schmidt, Astrid M. Z.; Gilbert, M. Thomas P.; Hansen, Anders J.; Orlando, Ludovic; Willerslev, Eske

    2015-01-01

    DNA obtained from environmental samples such as sediments, ice or water (environmental DNA, eDNA), represents an important source of information on past and present biodiversity. It has revealed an ancient forest in Greenland, extended by several thousand years the survival dates for mainland woolly mammoth in Alaska, and pushed back the dates for spruce survival in Scandinavian ice-free refugia during the last glaciation. More recently, eDNA was used to uncover the past 50 000 years of vegetation history in the Arctic, revealing massive vegetation turnover at the Pleistocene/Holocene transition, with implications for the extinction of megafauna. Furthermore, eDNA can reflect the biodiversity of extant flora and fauna, both qualitatively and quantitatively, allowing detection of rare species. As such, trace studies of plant and vertebrate DNA in the environment have revolutionized our knowledge of biogeography. However, the approach remains marred by biases related to DNA behaviour in environmental settings, incomplete reference databases and false positive results due to contamination. We provide a review of the field. PMID:25487334

  6. How We Make DNA Origami.

    Science.gov (United States)

    Wagenbauer, Klaus F; Engelhardt, Floris A S; Stahl, Evi; Hechtl, Vera K; Stömmer, Pierre; Seebacher, Fabian; Meregalli, Letizia; Ketterer, Philip; Gerling, Thomas; Dietz, Hendrik

    2017-10-05

    DNA origami has attracted substantial attention since its invention ten years ago, due to the seemingly infinite possibilities that it affords for creating customized nanoscale objects. Although the basic concept of DNA origami is easy to understand, using custom DNA origami in practical applications requires detailed know-how for designing and producing the particles with sufficient quality and for preparing them at appropriate concentrations with the necessary degree of purity in custom environments. Such know-how is not readily available for newcomers to the field, thus slowing down the rate at which new applications outside the field of DNA nanotechnology may emerge. To foster faster progress, we share in this article the experience in making and preparing DNA origami that we have accumulated over recent years. We discuss design solutions for creating advanced structural motifs including corners and various types of hinges that expand the design space for the more rigid multilayer DNA origami and provide guidelines for preventing undesired aggregation and on how to induce specific oligomerization of multiple DNA origami building blocks. In addition, we provide detailed protocols and discuss the expected results for five key methods that allow efficient and damage-free preparation of DNA origami. These methods are agarose-gel purification, filtration through molecular cut-off membranes, PEG precipitation, size-exclusion chromatography, and ultracentrifugation-based sedimentation. The guide for creating advanced design motifs and the detailed protocols with their experimental characterization that we describe here should lower the barrier for researchers to accomplish the full DNA origami production workflow. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. DNA-Controlled Assembly of Soft Nanoparticles

    DEFF Research Database (Denmark)

    Vogel, Stefan

    2015-01-01

    This book covers the emerging topic of DNA nanotechnology and DNA supramolecular chemistry in its broader sense. By taking DNA out of its biological role, this biomolecule has become a very versatile building block in materials chemistry, supramolecular chemistry and bio-nanotechnology. Many nove......-covalent systems, DNA origami, DNA based switches, DNA machines, and alternative structures and templates. This broad coverage is very appealing since it combines both the synthesis of modified DNA as well as designer concepts to successfully plan and make DNA nanostructures....

  8. Radicals of DNA and DNA nucleotides generated by ionising radiation

    International Nuclear Information System (INIS)

    Przybytniak, G.

    2004-01-01

    A first stage of cell processes leading to DNA damage of initiated by radical reactions. In a model system such transformations were generated by ionising radiation which involves production of electron loss and electron gain centers of the substrate and radical formation. Using cryogenic ESR spectroscopy it was found that the DNA nucleotides, which convert to radical anions upon electron capture undergo the separation of unpaired spin and charge due to protonation. Circular and linear dichroism studies enabled to conclude that iron ions(III) induce strong changes in the DNA helical structure indicating their coordination with nitrogen bases. The repair of DNA radicals produced via radiolytic oxidation, i.e. the guanine radical cation and the allyl type radical of thymine, is possible at elevated temperatures due to the involvement of sulphydryl groups. The influence of the thiol charge is then limited

  9. Environmental influences on DNA curvature

    DEFF Research Database (Denmark)

    Ussery, David; Higgins, C.F.; Bolshoy, A.

    1999-01-01

    DNA curvature plays an important role in many biological processes. To study environmentalinfluences on DNA curvature we compared the anomalous migration on polyacrylamide gels ofligation ladders of 11 specifically-designed oligonucleotides. At low temperatures (25 degreesC and below) most......, whilst spermine enhanced theanomalous migration of a different set of sequences. Sequences with a GGC motif exhibitedgreater curvature than predicted by the presently-used angles for the nearest-neighbour wedgemodel and are especially sensitive to Mg2+. The data have implications for models...... for DNAcurvature and for environmentally-sensitive DNA conformations in the regulation of geneexpression....

  10. Normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1997-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

  11. DNA Probe for Lactobacillus delbrueckii

    Science.gov (United States)

    Delley, Michèle; Mollet, Beat; Hottinger, Herbert

    1990-01-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-32P-labeled DNA probe. Images PMID:16348233

  12. DNA Uptake by Transformable Bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, Sanford A.

    1999-03-31

    The various processes of DNA uptake by cells can be categorized as: viral DNA entry, conjugation, or transformation. Within each category, a variety of mechanisms have been found. However, considerable similarities occur among the different mechanisms of conjugation and, especially, transformation. All of these natural mechanisms of DNA transfer are quite elaborate and involve multiple protein components, as the case may be, of the virus, the donor cell, and the recipient cell. The mechanisms of viral infection and conjugation will be discussed mainly with respect to their relevance to transformation.

  13. DNA UPTAKE BY TRANSFORMABLE BACTERIA

    Energy Technology Data Exchange (ETDEWEB)

    LACKS,S.A.

    1999-09-07

    The various processes of DNA uptake by cells can be categorized as: viral DNA entry, conjugation, or transformation. Within each category, a variety of mechanisms have been found. However, considerable similarities occur among the different mechanisms of conjugation and, especially, transformation. All of these natural mechanisms of DNA transfer are quite elaborate and involve multiple protein components, as the case may be, of the virus, the donor cell, and the recipient cell. The mechanisms of viral infection and conjugation will be discussed mainly with respect to their relevance to transformation.

  14. Recruitment of DNA methyltransferase I to DNA repair sites

    Science.gov (United States)

    Mortusewicz, Oliver; Schermelleh, Lothar; Walter, Joachim; Cardoso, M. Cristina; Leonhardt, Heinrich

    2005-01-01

    In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair. PMID:15956212

  15. Bacterial identification and subtyping using DNA microarray and DNA sequencing.

    Science.gov (United States)

    Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

    2012-01-01

    The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

  16. Comparison of five DNA quantification methods

    DEFF Research Database (Denmark)

    Nielsen, Karsten; Mogensen, Helle Smidt; Hedman, Johannes

    2008-01-01

    Six commercial preparations of human genomic DNA were quantified using five quantification methods: UV spectrometry, SYBR-Green dye staining, slot blot hybridization with the probe D17Z1, Quantifiler Human DNA Quantification kit and RB1 rt-PCR. All methods measured higher DNA concentrations than...... Quantification kit in two experiments. The measured DNA concentrations with Quantifiler were 125 and 160% higher than expected based on the manufacturers' information. When the Quantifiler human DNA standard (Raji cell line) was replaced by the commercial human DNA preparation G147A (Promega) to generate the DNA...... standard curve in the Quantifiler Human DNA Quantification kit, the DNA quantification results of the human DNA preparations were 31% higher than expected based on the manufacturers' information. The results indicate a calibration problem with the Quantifiler human DNA standard for its use...

  17. Synchronization of DNA array replication kinetics

    Science.gov (United States)

    Manturov, Alexey O.; Grigoryev, Anton V.

    2016-04-01

    In the present work we discuss the features of the DNA replication kinetics at the case of multiplicity of simultaneously elongated DNA fragments. The interaction between replicated DNA fragments is carried out by free protons that appears at the every nucleotide attachment at the free end of elongated DNA fragment. So there is feedback between free protons concentration and DNA-polymerase activity that appears as elongation rate dependence. We develop the numerical model based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) for DNA elongation process with conditions pointed above and we study the possibility of the DNA polymerases movement synchronization. The results obtained numerically can be useful for DNA polymerase movement detection and visualization of the elongation process in the case of massive DNA replication, eg, under PCR condition or for DNA "sequencing by synthesis" sequencing devices evaluation.

  18. DNA repair deficiency in neurodegeneration

    DEFF Research Database (Denmark)

    Jeppesen, Dennis Kjølhede; Bohr, Vilhelm A; Stevnsner, Tinna V.

    2011-01-01

    Deficiency in repair of nuclear and mitochondrial DNA damage has been linked to several neurodegenerative disorders. Many recent experimental results indicate that the post-mitotic neurons are particularly prone to accumulation of unrepaired DNA lesions potentially leading to progressive...... neurodegeneration. Nucleotide excision repair is the cellular pathway responsible for removing helix-distorting DNA damage and deficiency in such repair is found in a number of diseases with neurodegenerative phenotypes, including Xeroderma Pigmentosum and Cockayne syndrome. The main pathway for repairing oxidative...... base lesions is base excision repair, and such repair is crucial for neurons given their high rates of oxygen metabolism. Mismatch repair corrects base mispairs generated during replication and evidence indicates that oxidative DNA damage can cause this pathway to expand trinucleotide repeats, thereby...

  19. Sorting fluorescent nanocrystals with DNA

    Energy Technology Data Exchange (ETDEWEB)

    Gerion, Daniele; Parak, Wolfgang J.; Williams, Shara C.; Zanchet, Daniela; Micheel, Christine M.; Alivisatos, A. Paul

    2001-12-10

    Semiconductor nanocrystals with narrow and tunable fluorescence are covalently linked to oligonucleotides. These biocompounds retain the properties of both nanocrystals and DNA. Therefore, different sequences of DNA can be coded with nanocrystals and still preserve their ability to hybridize to their complements. We report the case where four different sequences of DNA are linked to four nanocrystal samples having different colors of emission in the range of 530-640 nm. When the DNA-nanocrystal conjugates are mixed together, it is possible to sort each type of nanoparticle using hybridization on a defined micrometer -size surface containing the complementary oligonucleotide. Detection of sorting requires only a single excitation source and an epifluorescence microscope. The possibility of directing fluorescent nanocrystals towards specific biological targets and detecting them, combined with their superior photo-stability compared to organic dyes, opens the way to improved biolabeling experiments, such as gene mapping on a nanometer scale or multicolor microarray analysis.

  20. DNA photoprotection conveyed by sunscreen.

    Science.gov (United States)

    DeHaven, Charlene; Hayden, Patrick J; Armento, Alexander; Oldach, Jonathan

    2014-06-01

    Skin photoaging is the consequence of solar UV exposure, and DNA damage is an important part of this process. The objective of the current work was to demonstrate that in vitro skin models can be utilized to confirm that DNA damage protection is provided by sunscreens. Skin equivalents were exposed to full-spectrum UV light administered with a standard research solar simulator with and without pre-application of sunscreen. Cyclopyrimidine dimer (CPD) and sunburn cell (SBC) formation as well as CPD quantitation were evaluated to determine DNA damage protection provided by the sunscreen. Marked decreases in both CPDs and SBCs were observed when sunscreen was applied prior to UV exposure. Sunscreen application prior to full-spectrum solar UV exposure protects DNA from photodamage measured by CPD and SBC formation. This can be expected to lessen the risk of photoaging and malignant transformation. © 2014 Wiley Periodicals, Inc.

  1. DNA vaccines for aquacultured fish

    DEFF Research Database (Denmark)

    Lorenzen, Niels; LaPatra, S.E.

    2005-01-01

    of licensing and public acceptance of the technology. The potential benefits of DNA vaccines for farmed fish include improved animal welfare, reduced environmental impacts of aquaculture activities, increased food quality and quantity, and more sustainable production. Testing under commercial production......Deoxyribonucleic acid (DNA) vaccination is based on the administration of the gene encoding the vaccine antigen, rather than the antigen itself. Subsequent expression of the antigen by cells in the vaccinated hosts triggers the host immune system. Among the many experimental DNA vaccines tested...... in various animal species as well as in humans, the vaccines against rhabdovirus diseases in fish have given some of the most promising results. A single intramuscular (IM) injection of microgram amounts of DNA induces rapid and long-lasting protection in farmed salmonids against economically important...

  2. Mammalian DNA Repair. Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Wood, Richard D.

    2003-01-24

    The Gordon Research Conference (GRC) on Mammalian DNA Repair was held at Harbortown Resort, Ventura Beach, CA. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  3. (UVB)-induced DNA damage

    African Journals Online (AJOL)

    Jane

    2011-08-17

    dependent cytogenetic lesions were assessed by the micronucleus test (MNT). It was found that POE effectively reduced the extent of DNA breakages and cytogenetic lesions upon exposure to UVB (erythemal ultraviolet (EUV);.

  4. Mitogenomic analyses from ancient DNA

    DEFF Research Database (Denmark)

    Paijmans, Johanna L. A.; Gilbert, Tom; Hofreiter, Michael

    2013-01-01

    The analysis of ancient DNA is playing an increasingly important role in conservation genetic, phylogenetic and population genetic analyses, as it allows incorporating extinct species into DNA sequence trees and adds time depth to population genetics studies. For many years, these types of DNA...... analyses (whether using modern or ancient DNA) were largely restricted to the analysis of short fragments of the mitochondrial genome. However, due to many technological advances during the past decade, a growing number of studies have explored the power of complete mitochondrial genome sequences...... yielded major progress with regard to both the phylogenetic positions of extinct species, as well as resolving population genetics questions in both extinct and extant species....

  5. Integrated Sensing Using DNA Nanoarchitectures

    Science.gov (United States)

    2014-05-20

    Norton. Thiolated Dendrimers as Multi-Point Binding Headgroups for DNA Immobilization on Gold, Langmuir, (10 2011): 0. doi: 10.1021/la202444s...flat mica surface, the structure is planar (it is conformal, lacking rigidity as a 2 nm thick polymer sheet. The simulated structure is shown in...Morris, John R., and Norton, Michael L.; Thiolated Dendrimers as Multi-Point Binding Headgroups for DNA Immobilization on Gold, Langmuir, 27(20

  6. LET-effects in DNA

    International Nuclear Information System (INIS)

    Kraft, G.; Taucher-Scholz, G.; Heilmann, J.

    1994-11-01

    In this contribution, an introductory view on the physical properties of ions is given and the cellular response to high LET radiation is summarized. Then the measurements of strand break induction of DNA in solution and in intracellular DNA are reported and compared to cell survival. The possibility of changes in the quality of the lesions is discussed and finally the present status of model calculations in comparison to the experiments is given. (orig./HSI)

  7. DNA Binding Hydroxyl Radical Probes

    OpenAIRE

    Tang, Vicky J; Konigsfeld, Katie M; Aguilera, Joe A; Milligan, Jamie R

    2012-01-01

    The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different c...

  8. DNA probe for lactobacillus delbrueckii

    Energy Technology Data Exchange (ETDEWEB)

    Delley, M.; Mollet, B.; Hottinger, H. (Nestle Research Centre, Lausanne (Switzerland))

    1990-06-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an {alpha}-{sup 32}P-labeled probe.

  9. DNA probe for lactobacillus delbrueckii

    International Nuclear Information System (INIS)

    Delley, M.; Mollet, B.; Hottinger, H.

    1990-01-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α- 32 P-labeled probe

  10. DNA Probe for Lactobacillus delbrueckii

    OpenAIRE

    Delley, Michèle; Mollet, Beat; Hottinger, Herbert

    1990-01-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-32P-l...

  11. DNA extraction from herbarium specimens.

    Science.gov (United States)

    Drábková, Lenka Záveská

    2014-01-01

    With the expansion of molecular techniques, the historical collections have become widely used. Studying plant DNA using modern molecular techniques such as DNA sequencing plays an important role in understanding evolutionary relationships, identification through DNA barcoding, conservation status, and many other aspects of plant biology. Enormous herbarium collections are an important source of material especially for specimens from areas difficult to access or from taxa that are now extinct. The ability to utilize these specimens greatly enhances the research. However, the process of extracting DNA from herbarium specimens is often fraught with difficulty related to such variables as plant chemistry, drying method of the specimen, and chemical treatment of the specimen. Although many methods have been developed for extraction of DNA from herbarium specimens, the most frequently used are modified CTAB and DNeasy Plant Mini Kit protocols. Nine selected protocols in this chapter have been successfully used for high-quality DNA extraction from different kinds of plant herbarium tissues. These methods differ primarily with respect to their requirements for input material (from algae to vascular plants), type of the plant tissue (leaves with incrustations, sclerenchyma strands, mucilaginous tissues, needles, seeds), and further possible applications (PCR-based methods or microsatellites, AFLP).

  12. DNA recognition by synthetic constructs.

    Science.gov (United States)

    Pazos, Elena; Mosquera, Jesús; Vázquez, M Eugenio; Mascareñas, José L

    2011-09-05

    The interaction of transcription factors with specific DNA sites is key for the regulation of gene expression. Despite the availability of a large body of structural data on protein-DNA complexes, we are still far from fully understanding the molecular and biophysical bases underlying such interactions. Therefore, the development of non-natural agents that can reproduce the DNA-recognition properties of natural transcription factors remains a major and challenging goal in chemical biology. In this review we summarize the basics of double-stranded DNA recognition by transcription factors, and describe recent developments in the design and preparation of synthetic DNA binders. We mainly focus on synthetic peptides that have been designed by following the DNA interaction of natural proteins, and we discuss how the tools of organic synthesis can be used to make artificial constructs equipped with functionalities that introduce additional properties to the recognition process, such as sensing and controllability. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Fanconi anemia and DNA repair.

    Science.gov (United States)

    Grompe, M; D'Andrea, A

    2001-10-01

    Fanconi anemia (FA) is an autosomal recessive disorder caused by defects in at least eight distinct genes FANCA, B, C, D1, D2, E, F and G. The clinical phenotype of all FA complementation groups is similar and is characterized by progressive bone marrow failure, cancer proneness and typical birth defects. The principal cellular phenotype is hypersensitivity to DNA damage, particularly interstrand DNA crosslinks. The FA proteins constitute a multiprotein pathway whose precise biochemical function(s) remain unknown. Five of the FA proteins (FANCA, C, E, F and G) interact in a nuclear complex upstream of FANCD2. FANCB and FANCD1 have not yet been cloned, but it is likely that FANCB is part of the nuclear complex and that FANCD1 acts downstream of FANCD2. The FA nuclear complex regulates the mono-ubiquitination of FANCD2 in response to DNA damage, resulting in targeting of this protein into nuclear foci. These foci also contain BRCA1 and other DNA damage response proteins. In male meiosis, FANCD2 also co-localizes with BRCA1 at synaptonemal complexes. Together, these data suggest that the FA pathway functions primarily as a DNA damage response system, although its exact role (direct involvement in DNA repair versus indirect, facilitating role) has not yet been defined.

  14. DNA-scaffolded nanoparticle structures

    Energy Technology Data Exchange (ETDEWEB)

    Hoegberg, Bjoern; Olin, Haakan [Department of Engineering Physics and Mathematics, Mid Sweden University, SE-851 70 Sundsvall, Sweden (Sweden)

    2007-03-15

    DNA self-assembly is a powerful route to the production of very small, complex structures. When used in combination with nanoparticles it is likely to become a key technology in the production of nanoelectronics in the future. Previously, demonstrated nanoparticle assemblies have mainly been periodic and highly symmetric arrays, unsuited as building blocks for any complex circuits. With the invention of DNA-scaffolded origami reported earlier this year (Rothemund P W K 2006 Nature 440 (7082) 297-302), a new route to complex nanostructures using DNA has been opened. Here, we give a short review of the field and present the current status of our experiments were DNA origami is used in conjunction with nanoparticles. Gold nanoparticles are functionalized with thiolated single stranded DNA. Strands that are complementary to the gold particle strands can be positioned on the self-assembled DNA-structure in arbitrary patterns. This property should allow an accurate positioning of the particles by letting them hybridize on the lattice. We report on our recent experiments on this system and discuss open problems and future applications.

  15. DNA-scaffolded nanoparticle structures

    International Nuclear Information System (INIS)

    Hoegberg, Bjoern; Olin, Haakan

    2007-01-01

    DNA self-assembly is a powerful route to the production of very small, complex structures. When used in combination with nanoparticles it is likely to become a key technology in the production of nanoelectronics in the future. Previously, demonstrated nanoparticle assemblies have mainly been periodic and highly symmetric arrays, unsuited as building blocks for any complex circuits. With the invention of DNA-scaffolded origami reported earlier this year (Rothemund P W K 2006 Nature 440 (7082) 297-302), a new route to complex nanostructures using DNA has been opened. Here, we give a short review of the field and present the current status of our experiments were DNA origami is used in conjunction with nanoparticles. Gold nanoparticles are functionalized with thiolated single stranded DNA. Strands that are complementary to the gold particle strands can be positioned on the self-assembled DNA-structure in arbitrary patterns. This property should allow an accurate positioning of the particles by letting them hybridize on the lattice. We report on our recent experiments on this system and discuss open problems and future applications

  16. Mechanical design of DNA nanostructures

    Science.gov (United States)

    Castro, Carlos E.; Su, Hai-Jun; Marras, Alexander E.; Zhou, Lifeng; Johnson, Joshua

    2015-03-01

    Structural DNA nanotechnology is a rapidly emerging field that has demonstrated great potential for applications such as single molecule sensing, drug delivery, and templating molecular components. As the applications of DNA nanotechnology expand, a consideration of their mechanical behavior is becoming essential to understand how these structures will respond to physical interactions. This review considers three major avenues of recent progress in this area: (1) measuring and designing mechanical properties of DNA nanostructures, (2) designing complex nanostructures based on imposed mechanical stresses, and (3) designing and controlling structurally dynamic nanostructures. This work has laid the foundation for mechanically active nanomachines that can generate, transmit, and respond to physical cues in molecular systems.Structural DNA nanotechnology is a rapidly emerging field that has demonstrated great potential for applications such as single molecule sensing, drug delivery, and templating molecular components. As the applications of DNA nanotechnology expand, a consideration of their mechanical behavior is becoming essential to understand how these structures will respond to physical interactions. This review considers three major avenues of recent progress in this area: (1) measuring and designing mechanical properties of DNA nanostructures, (2) designing complex nanostructures based on imposed mechanical stresses, and (3) designing and controlling structurally dynamic nanostructures. This work has laid the foundation for mechanically active nanomachines that can generate, transmit, and respond to physical cues in molecular systems. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr07153k

  17. Satellite DNA: An Evolving Topic.

    Science.gov (United States)

    Garrido-Ramos, Manuel A

    2017-09-18

    Satellite DNA represents one of the most fascinating parts of the repetitive fraction of the eukaryotic genome. Since the discovery of highly repetitive tandem DNA in the 1960s, a lot of literature has extensively covered various topics related to the structure, organization, function, and evolution of such sequences. Today, with the advent of genomic tools, the study of satellite DNA has regained a great interest. Thus, Next-Generation Sequencing (NGS), together with high-throughput in silico analysis of the information contained in NGS reads, has revolutionized the analysis of the repetitive fraction of the eukaryotic genomes. The whole of the historical and current approaches to the topic gives us a broad view of the function and evolution of satellite DNA and its role in chromosomal evolution. Currently, we have extensive information on the molecular, chromosomal, biological, and population factors that affect the evolutionary fate of satellite DNA, knowledge that gives rise to a series of hypotheses that get on well with each other about the origin, spreading, and evolution of satellite DNA. In this paper, I review these hypotheses from a methodological, conceptual, and historical perspective and frame them in the context of chromosomal organization and evolution.

  18. DNA Damage and Pulmonary Hypertension

    Science.gov (United States)

    Ranchoux, Benoît; Meloche, Jolyane; Paulin, Roxane; Boucherat, Olivier; Provencher, Steeve; Bonnet, Sébastien

    2016-01-01

    Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure over 25 mmHg at rest and is diagnosed by right heart catheterization. Among the different groups of PH, pulmonary arterial hypertension (PAH) is characterized by a progressive obstruction of distal pulmonary arteries, related to endothelial cell dysfunction and vascular cell proliferation, which leads to an increased pulmonary vascular resistance, right ventricular hypertrophy, and right heart failure. Although the primary trigger of PAH remains unknown, oxidative stress and inflammation have been shown to play a key role in the development and progression of vascular remodeling. These factors are known to increase DNA damage that might favor the emergence of the proliferative and apoptosis-resistant phenotype observed in PAH vascular cells. High levels of DNA damage were reported to occur in PAH lungs and remodeled arteries as well as in animal models of PH. Moreover, recent studies have demonstrated that impaired DNA-response mechanisms may lead to an increased mutagen sensitivity in PAH patients. Finally, PAH was linked with decreased breast cancer 1 protein (BRCA1) and DNA topoisomerase 2-binding protein 1 (TopBP1) expression, both involved in maintaining genome integrity. This review aims to provide an overview of recent evidence of DNA damage and DNA repair deficiency and their implication in PAH pathogenesis. PMID:27338373

  19. DNA testing in homicide investigations.

    Science.gov (United States)

    Prahlow, Joseph A; Cameron, Thomas; Arendt, Alexander; Cornelis, Kenneth; Bontrager, Anthony; Suth, Michael S; Black, Lisa; Tobey, Rebbecca; Pollock, Sharon; Stur, Shawn; Cotter, Kenneth; Gabrielse, Joel

    2017-10-01

    Objectives With the widespread use of DNA testing, police, death investigators, and attorneys need to be aware of the capabilities of this technology. This review provides an overview of scenarios where DNA evidence has played a major role in homicide investigations in order to highlight important educational issues for police, death investigators, forensic pathologists, and attorneys. Methods This was a nonrandom, observational, retrospective study. Data were obtained from the collective files of the authors from casework during a 15-year period, from 2000 through 2014. Results A series of nine scenarios, encompassing 11 deaths, is presented from the standpoint of the police and death investigation, the forensic pathology autopsy performance, the subsequent DNA testing of evidence, and, ultimately, the final adjudication of cases. Details of each case are presented, along with a discussion that focuses on important aspects of sample collection for potential DNA testing, especially at the crime scene and the autopsy. The presentation highlights the diversity of case and evidence types in which DNA testing played a valuable role in the successful prosecution of the case. Conclusions By highlighting homicides where DNA testing contributed to the successful adjudication of cases, police, death investigators, forensic pathologists, and attorneys will be better informed regarding the types of evidence and situations where such testing is of potential value.

  20. DNA damage in plant herbarium tissue.

    NARCIS (Netherlands)

    Staats, M.; Cuenca, A.; Richardson, J.E.; Ginkel, R.V.; Petersen, G.; Seberg, O.; Bakker, F.T.

    2011-01-01

    Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of

  1. Alternative end-joining of DNA breaks

    NARCIS (Netherlands)

    Schendel, Robin van

    2016-01-01

    DNA is arguably the most important molecule found in any organism, as it contains all information to perform cellular functions and enables continuity of species. It is continuously exposed to DNA-damaging agents both from endogenous and exogenous sources. To protect DNA against these sources of DNA

  2. Chemically modified DNA : methods and applications

    NARCIS (Netherlands)

    Gjonaj, Lorina

    2013-01-01

    Over het toepassen van DNA in de chemie DNA is een fascinerend biopolymeer dat gemakkelijk gemanipuleerd kan worden. Doel van het onderzoek van Lorina Gjonaj was het ontwikkelen van nieuwe methodologie om DNA chemisch te modificeren. Verschillende functionaliteiten zijn aan DNA gekoppeld. Deze

  3. DNA Fingerprinting in a Forensic Teaching Experiment

    Science.gov (United States)

    Wagoner, Stacy A.; Carlson, Kimberly A.

    2008-01-01

    This article presents an experiment designed to provide students, in a classroom laboratory setting, a hands-on demonstration of the steps used in DNA forensic analysis by performing DNA extraction, DNA fingerprinting, and statistical analysis of the data. This experiment demonstrates how DNA fingerprinting is performed and how long it takes. It…

  4. Fetal DNA: strategies for optimal recovery

    NARCIS (Netherlands)

    Legler, Tobias J.; Heermann, Klaus-Hinrich; Liu, Zhong; Soussan, Aicha Ait; van der Schoot, C. Ellen

    2008-01-01

    For fetal DNA extraction, in principle each DNA extraction method can be used; however, because most methods have been optimized for genomic DNA from leucocytes, we describe here the methods that have been optimized for the extraction of fetal DNA from maternal plasma and validated for this purpose

  5. Extracellular DNA metabolism in Haloferax volcanii

    Directory of Open Access Journals (Sweden)

    Scott eChimileski

    2014-02-01

    Full Text Available Extracellular DNA is found in all environments and is a dynamic component of the micro-bial ecosystem. Microbial cells produce and interact with extracellular DNA through many endogenous mechanisms. Extracellular DNA is processed and internalized for use as genetic information and as a major source of macronutrients, and plays several key roles within prokaryotic biofilms. Hypersaline sites contain some of the highest extracellular DNA con-centrations measured in nature–a potential rich source of carbon, nitrogen and phosphorus for halophilic microorganisms. We conducted DNA growth studies for the halophilic archaeon Haloferax volcanii DS2 and show that this model Halobacteriales strain is capable of using exogenous double-stranded DNA as a nutrient. Further experiments with varying medium composition, DNA concentration and DNA types revealed that DNA is utilized primarily as a phosphorus source, that growth on DNA is concentration-dependent and that DNA isolated from different sources is metabolized selectively, with a bias against highly divergent methylated DNA sources. Additionally, fluorescence microscopy experiments showed that labeled DNA colocalized with Haloferax volcanii cells. The gene Hvo_1477 was also identified using a comparative genomic approach as a factor likely to be involved in extracellular DNA processing at the cell surface, and deletion of Hvo_1477 created an H. volcanii strain deficient in its ability to grow on extracellular DNA. Widespread distribution of Hvo_1477 homologs in archaea suggests metabolism of extracellular DNA may be of broad ecological and physiological relevance in this domain of life.

  6. Disinfection of Vegetative Cells of Bacillus anthracis

    Science.gov (United States)

    2016-03-01

    Peterson, A.; Donlan, R.M.; Arduino , M.J. Chlorine Inactivation of Bacterial Select Agents. Appl. and Environ. Microbial. 2005, 71, 5669–5689...Rose, L.J.; Rice, E.W.; Hodges, L.; Peterson, A.; Arduino , M.J. 2007. Monochloramine Inactivation of Bacterial Select Agents. Appl. and Environ

  7. DNA polymerase beta participates in mitochondrial DNA repair

    DEFF Research Database (Denmark)

    Sykora, P; Kanno, S; Akbari, M

    2017-01-01

    We have detected DNA polymerase beta (Polβ), known as a key nuclear base excision repair (BER) protein, in mitochondrial protein extracts derived from mammalian tissue and cells. Manipulation of the N-terminal sequence affected the amount of Polβ in the mitochondria. Using Polβ fragments, mitocho......We have detected DNA polymerase beta (Polβ), known as a key nuclear base excision repair (BER) protein, in mitochondrial protein extracts derived from mammalian tissue and cells. Manipulation of the N-terminal sequence affected the amount of Polβ in the mitochondria. Using Polβ fragments......, mitochondrial-specific protein partners were identified, with the interactors mainly functioning in DNA maintenance and mitochondrial import. Of particular interest was the identification of the proteins TWINKLE, SSBP1 and TFAM, all of which are mitochondria specific DNA effectors and are known to function...... in the nucleoid. Polβ directly interacted with, and influenced the activity of, the mitochondrial helicase TWINKLE. Human kidney cells with Polβ knock-out (KO) had higher endogenous mtDNA damage. Mitochondrial extracts derived from heterozygous Polβ mouse tissue and KO cells had lower nucleotide incorporation...

  8. Radiation damage to DNA in DNA-protein complexes.

    Science.gov (United States)

    Spotheim-Maurizot, M; Davídková, M

    2011-06-03

    The most aggressive product of water radiolysis, the hydroxyl (OH) radical, is responsible for the indirect effect of ionizing radiations on DNA in solution and aerobic conditions. According to radiolytic footprinting experiments, the resulting strand breaks and base modifications are inhomogeneously distributed along the DNA molecule irradiated free or bound to ligands (polyamines, thiols, proteins). A Monte-Carlo based model of simulation of the reaction of OH radicals with the macromolecules, called RADACK, allows calculating the relative probability of damage of each nucleotide of DNA irradiated alone or in complexes with proteins. RADACK calculations require the knowledge of the three dimensional structure of DNA and its complexes (determined by X-ray crystallography, NMR spectroscopy or molecular modeling). The confrontation of the calculated values with the results of the radiolytic footprinting experiments together with molecular modeling calculations show that: (1) the extent and location of the lesions are strongly dependent on the structure of DNA, which in turns is modulated by the base sequence and by the binding of proteins and (2) the regions in contact with the protein can be protected against the attack by the hydroxyl radicals via masking of the binding site and by scavenging of the radicals. 2011 Elsevier B.V. All rights reserved.

  9. JavaScript DNA translator: DNA-aligned protein translations.

    Science.gov (United States)

    Perry, William L

    2002-12-01

    There are many instances in molecular biology when it is necessary to identify ORFs in a DNA sequence. While programs exist for displaying protein translations in multiple ORFs in alignment with a DNA sequence, they are often expensive, exist as add-ons to software that must be purchased, or are only compatible with a particular operating system. JavaScript DNA Translator is a shareware application written in JavaScript, a scripting language interpreted by the Netscape Communicator and Internet Explorer Web browsers, which makes it compatible with several different operating systems. While the program uses a familiar Web page interface, it requires no connection to the Internet since calculations are performed on the user's own computer. The program analyzes one or multiple DNA sequences and generates translations in up to six reading frames aligned to a DNA sequence, in addition to displaying translations as separate sequences in FASTA format. ORFs within a reading frame can also be displayed as separate sequences. Flexible formatting options are provided, including the ability to hide ORFs below a minimum size specified by the user. The program is available free of charge at the BioTechniques Software Library (www.Biotechniques.com).

  10. Mechanisms for radiation damage in DNA

    International Nuclear Information System (INIS)

    Sevilla, M.D.

    1985-07-01

    Radiation damage to DNA results from the direct interaction of radiation with DNA where positive ions, electrons and excited states are formed in the DNA, and the indirect effect where radical species formed in the surrounding medium by the radiation attack the DNA. The primary mechanism proposed for radiation damage, by the direct effect, is that positive and negative ions formed within the DNA strand migrate through the stacked DNA bases. The ions can then recombine, react with the DNA bases most likely to react by protonation of the anion and deprotonation or hydroxylation of the cation or transfer out of the DNA chain to the surrounding histone protein. This work as aimed at understanding the possible reactions of the DNA base ion radicals, as well as their initial distribution in the DNA strand. 31 refs

  11. Radiation-induced electron migration along DNA

    International Nuclear Information System (INIS)

    Fuciarelli, A.F.; Sisk, E.C.; Miller, J.H.; Zimbrick, J.D.

    1994-04-01

    Radiation-induced electron migration along DNA is a mechanism by which randomly produced stochastic energy deposition events can lead to nonrandom types of damage along DNA manifested distal to the sites of the initial energy deposition. Electron migration along DNA is significantly influenced by the DNA base sequence and DNA conformation. Migration along 7 base pairs in oligonucleotides containing guanine bases was observed for oligonucleotides irradiated in solution which compares to average migration distances of 6 to 10 bases for Escherichia coli DNA irradiated in solution and 5.5 base pairs for Escherichia coli DNA irradiated in cells. Evidence also suggests that electron migration can occur preferentially in the 5' to 3' direction along DNA. Our continued efforts will provide information regarding the contribution of electron transfer along DNA to formation of locally multiply damaged sites created in DNA by exposure to ionizing radiation

  12. Cellular responses to environmental DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    1994-08-01

    This volume contains the proceedings of the conference entitled Cellular Responses to Environmental DNA Damage held in Banff,Alberta December 1--6, 1991. The conference addresses various aspects of DNA repair in sessions titled DNA repair; Basic Mechanisms; Lesions; Systems; Inducible Responses; Mutagenesis; Human Population Response Heterogeneity; Intragenomic DNA Repair Heterogeneity; DNA Repair Gene Cloning; Aging; Human Genetic Disease; and Carcinogenesis. Individual papers are represented as abstracts of about one page in length.

  13. Enzymatic Ligation of Large Biomolecules to DNA

    DEFF Research Database (Denmark)

    Sørensen, Rasmus Schøler; Okholm, Anders Hauge; Schaffert, David Henning

    2013-01-01

    The ability to synthesize, characterize, and manipulate DNA forms the foundation of a range of advanced disciplines including genomics, molecular biology, and biomolecular engineering. In particular for the latter field, DNA has proven useful as a structural or functional component in nanoscale s....... As a proof of principle, parallelly labeled oligonucleotides were used to produce nanopatterned DNA origami structures, demonstrating rapid and versatile incorporation of non-DNA components into DNA nanoarchitectures....

  14. DNA repair , cell repair and radiosensitivity

    International Nuclear Information System (INIS)

    Zhestyanikov, V.D.

    1983-01-01

    Data obtained in laboratory of radiation cytology and literature data testifying to a considerable role of DNA repair in cell sensitivity to radiation and chemical DNA-tropic agents have been considered. Data pointing to the probability of contribution of inducible repair of DNA into plant cells sensitivity to X-rays are obtained. Certain violations of DNA repair do not result in the increase of radiosensitivity. It is assumed that in the cases unknown mechanisms of DNA repair operate

  15. Repeated extraction of DNA from FTA cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Ferrero, Laura; Børsting, Claus

    2011-01-01

    Extraction of DNA using magnetic bead based techniques on automated DNA extraction instruments provides a fast, reliable and reproducible method for DNA extraction from various matrices. However, the yield of extracted DNA from FTA-cards is typically low. Here, we demonstrate that it is possible...... to repeatedly extract DNA from the processed FTA-disk. The method increases the yield from the nanogram range to the microgram range....

  16. Modeling the mechanical properties of DNA nanostructures.

    Science.gov (United States)

    Arbona, Jean Michel; Aimé, Jean-Pierre; Elezgaray, Juan

    2012-11-01

    We discuss generalizations of a previously published coarse-grained description [Mergell et al., Phys. Rev. E 68, 021911 (2003)] of double stranded DNA (dsDNA). The model is defined at the base-pair level and includes the electrostatic repulsion between neighbor helices. We show that the model reproduces mechanical and elastic properties of several DNA nanostructures (DNA origamis). We also show that electrostatic interactions are necessary to reproduce atomic force microscopy measurements on planar DNA origamis.

  17. [Single-molecule detection and characterization of DNA replication based on DNA origami].

    Science.gov (United States)

    Wang, Qi; Fan, Youjie; Li, Bin

    2014-08-01

    To investigate single-molecule detection and characterization of DNA replication. Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication. The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis. The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

  18. Complex DNA structures and structures of DNA complexes

    International Nuclear Information System (INIS)

    Chazin, W.J.; Carlstroem, G.; Shiow-Meei Chen; Miick, S.; Gomez-Paloma, L.; Smith, J.; Rydzewski, J.

    1994-01-01

    Complex DNA structures (for example, triplexes, quadruplexes, junctions) and DNA-ligand complexes are more difficult to study by NMR than standard DNA duplexes are because they have high molecular weights, show nonstandard or distorted local conformations, and exhibit large resonance linewidths and severe 1 H spectral overlap. These systems also tend to have limited solubility and may require specialized solution conditions to maintain favorable spectral characteristics, which adds to the spectroscopic difficulties. Furthermore, with more atoms in the system, both assignment and structure calculation become more challenging. In this article, we focus on demonstrating the current status of NMR studies of such systems and the limitations to further progress; we also indicate in what ways isotopic enrichment can be useful

  19. Complex DNA structures and structures of DNA complexes

    Energy Technology Data Exchange (ETDEWEB)

    Chazin, W.J.; Carlstroem, G.; Shiow-Meei Chen; Miick, S.; Gomez-Paloma, L.; Smith, J.; Rydzewski, J. [Scripps Research Institute, La Jolla, CA (United States)

    1994-12-01

    Complex DNA structures (for example, triplexes, quadruplexes, junctions) and DNA-ligand complexes are more difficult to study by NMR than standard DNA duplexes are because they have high molecular weights, show nonstandard or distorted local conformations, and exhibit large resonance linewidths and severe {sup 1}H spectral overlap. These systems also tend to have limited solubility and may require specialized solution conditions to maintain favorable spectral characteristics, which adds to the spectroscopic difficulties. Furthermore, with more atoms in the system, both assignment and structure calculation become more challenging. In this article, we focus on demonstrating the current status of NMR studies of such systems and the limitations to further progress; we also indicate in what ways isotopic enrichment can be useful.

  20. Scaffolded DNA Origami of a DNA Tetrahedron Molecular Container

    DEFF Research Database (Denmark)

    Ke, Yongang; Sharma, Jaswinder; Liu, Minghui

    2009-01-01

    We describe a strategy of scaffolded DNA origami to design and construct 3D molecular cages of tetrahedron geometry with inside volume closed by triangular faces. Each edge of the triangular face is ∼54 nm in dimension. The estimated total external volume and the internal cavity of the triangular...... pyramid are about 1.8 × 10-23 and 1.5 × 10-23 m3, respectively. Correct formation of the tetrahedron DNA cage was verified by gel electrophoresis, atomic force microscopy, transmission electron microscopy, and dynamic light scattering techniques....