Science.gov

Sample records for attenuating factor phage

  1. CTXϕ: Exploring new alternatives in host factor-mediated filamentous phage replications.

    PubMed

    Martínez, Eriel; Campos-Gómez, Javier; Barre, François-Xavier

    2016-01-01

    For a long time Ff phages from Escherichia coli provided the majority of the knowledge about the rolling circle replication mechanism of filamentous phages. Host factors involved in coliphages replication have been fully identified. Based on these studies, the function of Rep protein as the accessory helicase directly implicated in filamentous phage replication was considered a paradigm. We recently reported that the replication of some filamentous phages from Vibrio cholerae, including the cholera toxin phage CTXϕ, depended on the accessory helicase UvrD instead of Rep. We also identified HU protein as one of the host factors involved in CTXϕ and VGJϕ replication. The requirement of UvrD and HU for rolling circle replication was previously reported in some family of plasmids but had no precedent in filamentous phages. Here, we enrich the discussion of our results and present new preliminary data highlighting remarkable divergence in the lifestyle of filamentous phages. PMID:27607139

  2. UHF Radio Wave Attenuation Factor Database

    NASA Astrophysics Data System (ADS)

    Khomenko, S. I.; Kostina, V. L.; Mytsenko, I. M.; Roenko, A. N.

    2007-07-01

    As is known each sea-going vessel is equipped with navigation, communication and other radio engineering facilities that serve to secure the safety of navigation and are chiefly operated at UHF-wave band. In developing these systems and calculating the energy potential for a necessary coverage range one should be well aware of the radio signal attenuation processes on a propagation path. The key parameter of this path is the (radio) wave attenuation factor V and its distance dependence V(R). A diversity of factors influencing the radio signal attenuation over the oceanic expanses, especially well pronounced and quite stable tropospheric ducts, and the lack of experimental data were the compelling reasons why the researchers of the Institute for Radiophysics and Electronics, NASU, had spent many years on comprehensive radiophysical investigations carried out in different regions of the Atlantic, Indian, Arctic and Pacific Oceans. The experimental data obtained allow creating the database of radio wave attenuation factor V.

  3. The Genomes, Proteomes, and Structures of Three Novel Phages That Infect the Bacillus cereus Group and Carry Putative Virulence Factors

    PubMed Central

    Belnap, David M.; Jensen, Jordan D.; Mathis, Andrew D.; Prince, John T.; Merrill, Bryan D.; Burnett, Sandra H.; Breakwell, Donald P.

    2014-01-01

    ABSTRACT This article reports the results of studying three novel bacteriophages, JL, Shanette, and Basilisk, which infect the pathogen Bacillus cereus and carry genes that may contribute to its pathogenesis. We analyzed host range and superinfection ability, mapped their genomes, and characterized phage structure by mass spectrometry and transmission electron microscopy (TEM). The JL and Shanette genomes were 96% similar and contained 217 open reading frames (ORFs) and 220 ORFs, respectively, while Basilisk has an unrelated genome containing 138 ORFs. Mass spectrometry revealed 23 phage particle proteins for JL and 15 for Basilisk, while only 11 and 4, respectively, were predicted to be present by sequence analysis. Structural protein homology to well-characterized phages suggested that JL and Shanette were members of the family Myoviridae, which was confirmed by TEM. The third phage, Basilisk, was similar only to uncharacterized phages and is an unrelated siphovirus. Cryogenic electron microscopy of this novel phage revealed a T=9 icosahedral capsid structure with the major capsid protein (MCP) likely having the same fold as bacteriophage HK97 MCP despite the lack of sequence similarity. Several putative virulence factors were encoded by these phage genomes, including TerC and TerD involved in tellurium resistance. Host range analysis of all three phages supports genetic transfer of such factors within the B. cereus group, including B. cereus, B. anthracis, and B. thuringiensis. This study provides a basis for understanding these three phages and other related phages as well as their contributions to the pathogenicity of B. cereus group bacteria. IMPORTANCE The Bacillus cereus group of bacteria contains several human and plant pathogens, including B. cereus, B. anthracis, and B. thuringiensis. Phages are intimately linked to the evolution of their bacterial hosts and often provide virulence factors, making the study of B. cereus phages important to understanding

  4. Influence of Environmental Factors on Phage-Bacteria Interaction and on the Efficacy and Infectivity of Phage P100.

    PubMed

    Fister, Susanne; Robben, Christian; Witte, Anna K; Schoder, Dagmar; Wagner, Martin; Rossmanith, Peter

    2016-01-01

    When using bacteriophages to control food-borne bacteria in food production plants and processed food, it is crucial to consider that environmental conditions influence their stability. These conditions can also affect the physiological state of bacteria and consequently host-virus interaction and the effectiveness of the phage ability to reduce bacteria numbers. In this study we investigated the stability, binding, and replication capability of phage P100 and its efficacy to control Listeria monocytogenes under conditions typically encountered in dairy plants. The influences of SDS, Lutensol AO 7, salt, smear water, and different temperatures were investigated. Results indicate that phage P100 is stable and able to bind to the host under most conditions tested. Replication was dependent upon the growth of L. monocytogenes and efficacy was higher when bacterial growth was reduced by certain environmental conditions. In long-term experiments at different temperatures phages were initially able to reduce bacteria up to seven log10 units after 2 weeks at 4°C. However, thereafter, re-growth and development of phage-resistant L. monocytogenes isolates were encountered. PMID:27516757

  5. Synthesis of tumor necrosis factor α for use as a mirror-image phage display target.

    PubMed

    Petersen, Mark E; Jacobsen, Michael T; Kay, Michael S

    2016-06-21

    Tumor Necrosis Factor alpha (TNFα) is an inflammatory cytokine that plays a central role in the pathogenesis of chronic inflammatory disease. Here we describe the chemical synthesis of l-TNFα along with the mirror-image d-protein for use as a phage display target. The synthetic strategy utilized native chemical ligation and desulfurization to unite three peptide segments, followed by oxidative folding to assemble the 52 kDa homotrimeric protein. This synthesis represents the foundational step for discovering an inhibitory d-peptide with the potential to improve current anti-TNFα therapeutic strategies. PMID:27211891

  6. Profiling lethal factor interacting proteins from human stomach using T7 phage display screening.

    PubMed

    Cardona-Correa, Albin; Rios-Velazquez, Carlos

    2016-05-01

    The anthrax lethal factor (LF) is a zinc dependent metalloproteinase that cleaves the majority of mitogen-activated protein kinase kinases and a member of NOD-like receptor proteins, inducing cell apoptosis. Despite efforts to fully understand the Bacillus anthracis toxin components, the gastrointestinal (GI) anthrax mechanisms have not been fully elucidated. Previous studies demonstrated gastric ulceration, and a substantial bacterial growth rate in Peyer's patches. However, the complete molecular pathways of the disease that results in tissue damage by LF proteolytic activity remains unclear. In the present study, to identify the profile of the proteins potentially involved in GI anthrax, protein‑protein interactions were investigated using human stomach T7 phage display (T7PD) cDNA libraries. T7PD is a high throughput technique that allows the expression of cloned DNA sequences as peptides on the phage surface, enabling the selection and identification of protein ligands. A wild type and mutant LF (E687A) were used to differentiate interaction sites. A total of 124 clones were identified from 194 interacting‑phages, at both the DNA and protein level, by in silico analysis. Databases revealed that the selected candidates were proteins from different families including lipase, peptidase‑A1 and cation transport families, among others. Furthermore, individual T7PD candidates were tested against LF in order to detect their specificity to the target molecule, resulting in 10 LF‑interacting peptides. With a minimum concentration of LF for interaction at 1 µg/ml, the T7PD isolated pepsin A3 pre‑protein (PAP) demonstrated affinity to both types of LF. In addition, PAP was isolated in various lengths for the same protein, exhibiting common regions following PRALINE alignment. These findings will help elucidate and improve the understanding of the molecular pathogenesis of GI anthrax, and aid in the development of potential therapeutic agents. PMID

  7. Profiling lethal factor interacting proteins from human stomach using T7 phage display screening

    PubMed Central

    CARDONA-CORREA, ALBIN; RIOS-VELAZQUEZ, CARLOS

    2016-01-01

    The anthrax lethal factor (LF) is a zinc dependent metalloproteinase that cleaves the majority of mitogen-activated protein kinase kinases and a member of NOD-like receptor proteins, inducing cell apoptosis. Despite efforts to fully understand the Bacillus anthracis toxin components, the gastrointestinal (GI) anthrax mechanisms have not been fully elucidated. Previous studies demonstrated gastric ulceration, and a substantial bacterial growth rate in Peyer's patches. However, the complete molecular pathways of the disease that results in tissue damage by LF proteolytic activity remains unclear. In the present study, to identify the profile of the proteins potentially involved in GI anthrax, protein-protein interactions were investigated using human stomach T7 phage display (T7PD) cDNA libraries. T7PD is a high throughput technique that allows the expression of cloned DNA sequences as peptides on the phage surface, enabling the selection and identification of protein ligands. A wild type and mutant LF (E687A) were used to differentiate interaction sites. A total of 124 clones were identified from 194 interacting-phages, at both the DNA and protein level, by in silico analysis. Databases revealed that the selected candidates were proteins from different families including lipase, peptidase-A1 and cation transport families, among others. Furthermore, individual T7PD candidates were tested against LF in order to detect their specificity to the target molecule, resulting in 10 LF-interacting peptides. With a minimum concentration of LF for interaction at 1 μg/ml, the T7PD isolated pepsin A3 pre-protein (PAP) demonstrated affinity to both types of LF. In addition, PAP was isolated in various lengths for the same protein, exhibiting common regions following PRALINE alignment. These findings will help elucidate and improve the understanding of the molecular pathogenesis of GI anthrax, and aid in the development of potential therapeutic agents. PMID:27035230

  8. Damping factor estimation using spin wave attenuation in permalloy film

    SciTech Connect

    Manago, Takashi; Yamanoi, Kazuto; Kasai, Shinya; Mitani, Seiji

    2015-05-07

    Damping factor of a Permalloy (Py) thin film is estimated by using the magnetostatic spin wave propagation. The attenuation lengths are obtained by the dependence of the transmission intensity on the antenna distance, and decrease with increasing magnetic fields. The relationship between the attenuation length, damping factor, and external magnetic field is derived theoretically, and the damping factor was determined to be 0.0063 by fitting the magnetic field dependence of the attenuation length, using the derived equation. The obtained value is in good agreement with the general value of Py. Thus, this estimation method of the damping factor using spin waves attenuation can be useful tool for ferromagnetic thin films.

  9. Neutralisation of factor VIII inhibitors by anti-idiotypes isolated from phage-displayed libraries.

    PubMed

    Schmidt, Anja; Brettschneider, Kerstin; Kahle, Jörg; Orlowski, Aleksander; Becker-Peters, Karin; Stichel, Diana; Schulze, Jörg; Braner, Markus; Tampé, Robert; Schwabe, Dirk; Königs, Christoph

    2016-07-01

    Following replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated anti-idiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitor-specific, high-affinity anti-idiotypes can be isolated from phage-displayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations. PMID:27009573

  10. X-Ray Form Factor, Attenuation and Scattering Tables

    National Institute of Standards and Technology Data Gateway

    SRD 66 X-Ray Form Factor, Attenuation and Scattering Tables (Web, free access)   This database collects tables and graphs of the form factors, the photoabsorption cross section, and the total attenuation coefficient for any element (Z <= 92).

  11. Inhibition of transcription in Staphylococcus aureus by a primary sigma factor-binding polypeptide from phage G1.

    PubMed

    Dehbi, Mohammed; Moeck, Gregory; Arhin, Francis F; Bauda, Pascale; Bergeron, Dominique; Kwan, Tony; Liu, Jing; McCarty, John; Dubow, Michael; Pelletier, Jerry

    2009-06-01

    The primary sigma factor of Staphylococcus aureus, sigma(SA), regulates the transcription of many genes, including several essential genes, in this bacterium via specific recognition of exponential growth phase promoters. In this study, we report the existence of a novel staphylococcal phage G1-derived growth inhibitory polypeptide, referred to as G1ORF67, that interacts with sigma(SA) both in vivo and in vitro and regulates its activity. Delineation of the minimal domain of sigma(SA) that is required for its interaction with G1ORF67 as amino acids 294 to 360 near the carboxy terminus suggests that the G1 phage-encoded anti-sigma factor may occlude the -35 element recognition domain of sigma(SA). As would be predicted by this hypothesis, the G1ORF67 polypeptide abolished both RNA polymerase core-dependent binding of sigma(SA) to DNA and sigma(SA)-dependent transcription in vitro. While G1ORF67 profoundly inhibits transcription when expressed in S. aureus cells in mode of action studies, our finding that G1ORF67 was unable to inhibit transcription when expressed in Escherichia coli concurs with its inability to inhibit transcription by the E. coli holoenzyme in vitro. These features demonstrate the selectivity of G1ORF67 for S. aureus RNA polymerase. We predict that G1ORF67 is one of the central polypeptides in the phage G1 strategy to appropriate host RNA polymerase and redirect it to phage reproduction. PMID:19376864

  12. Phage Transduction.

    PubMed

    Goh, Shan

    2016-01-01

    Bacteriophages mediate horizontal gene transfer through a mechanism known as transduction. Phage transduction carried out in the laboratory involves a bacterial donor and a recipient, both of which are susceptible to infection by the phage of interest. Phage is propagated in the donor, concentrated, and exposed transiently to recipient at different multiplicity of infection ratios. Transductants are selected for the desired phenotype by culture on selective medium. Here we describe transduction of ermB conferring resistance to erythromycin by the C. difficile phage ϕC2. PMID:27507341

  13. Ketoconazole attenuates radiation-induction of tumor necrosis factor

    SciTech Connect

    Hallahan, D.E.; Virudachalam, S.; Kufe, D.W.; Weichselbaum, R.R.

    1994-07-01

    Previous work has demonstrated that inhibitors of phospholipase A2 attenuate ionizing radiation-induced arachidonic acid production, protein kinase C activation, and prevent subsequent induction of the tumor necrosis factor gene. Because arachidonic acid contributes to radiation-induced tumor necrosis factor expression, the authors analyzed the effects of agents which alter arachidonate metabolism on the regulation of this gene. Phospholipase A2 inhibitors quinicrine, bromphenyl bromide, and pentoxyfylline or the inhibitor of lipoxygenase (ketoconazole) or the inhibitor of cycloxygenase (indomethacine) were added to cell culture 1 h prior to irradiation. Radiation-induced tumor necrosis factor gene expression was attenuated by each of the phospholipase A2 inhibitors (quinicrine, bromphenylbromide, and pentoxyfylline). Furthermore, ketoconazole attenuated X ray induced tumor necrosis factor gene expression. Conversely, indomethacin enhanced tumor necrosis factor expression following irradiation. The finding that radiation-induced tumor necrosis factor gene expression was attenuated by ketoconazole suggests that the lipoxygenase pathway participates in signal transduction preceding tumor necrosis factor induction. Enhancement of tumor necrosis factor expression by indomethacin following irradiation suggests that prostaglandins produced by cyclooxygenase act as negative regulators of tumor necrosis factor expression. Inhibitors of tumor necrosis factor induction ameliorate acute and subacute sequelae of radiotherapy. The authors propose therefore, that ketoconazole may reduce acute radiation sequelae such as mucositis and esophagitis through a reduction in tumor necrosis factor induction or inhibition of phospholipase A2 in addition to its antifungal activity. 25 refs., 2 figs.

  14. Binding-induced Stabilization and Assembly of the Phage P22 Tail Accessory Factor gp4

    SciTech Connect

    Olia,A.; Al-Bassam, J.; Winn-Stapley, D.; Joss, L.; Casjens, S.; Cingolani, G.

    2006-01-01

    To infect and replicate, bacteriophage P22 injects its 43 kbp genome across the cell wall of Salmonella enterica serovar Typhimurium. The attachment of phage P22 to the host cell as well as the injection of the viral DNA into the host is mediated by the virion's tail complex. This 2.8 MDa molecular machine is formed by five proteins, which include the portal protein gp1, the adhesion tailspike protein gp9, and three tail accessory factors: gp4, gp10, gp26. We have isolated the tail accessory factor gp4 and characterized its structure and binding interactions with portal protein. Interestingly, gp4 exists in solution as a monomer, which displays an exceedingly low structural stability (T{sub m} 34 {sup o}C). Unfolded gp4 is prone to aggregation within a narrow range of temperatures both in vitro and in Salmonella extracts. In the virion the thermal unfolding of gp4 is prevented by the interaction with the dodecameric portal protein, which stabilizes the structure of gp4 and suppresses unfolded gp4 from irreversibly aggregating in the Salmonella milieu. The structural stabilization of gp4 is accompanied by the concomitant oligomerization of the protein to form a ring of 12 subunits bound to the lower end of the portal ring. The interaction of gp4 with portal protein is complex and likely involves the distinct binding of two non-equivalent sets of six gp4 proteins. Binding of the first set of six gp4 equivalents to dodecameric portal protein yields a gp(1){sub 12}:gp(4){sub 6} assembly intermediate, which is stably populated at 30 {sup o}C and can be resolved by native gel electrophoresis. The final product of the assembly reaction is a bi-dodecameric gp(1){sub 12}:gp(4){sub 12} complex, which appears hollow by electron microscopy, suggesting that gp4 does not physically plug the DNA entry/exit channel, but acts as a structural adaptor for the other tail accessory factors: gp10 and gp26.

  15. Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy.

    PubMed

    Lai, Yan-Da; Wu, Yen-Yu; Tsai, Yi-Jiue; Tsai, Yi-San; Lin, Yu-Ying; Lai, Szu-Liang; Huang, Chao-Yang; Lok, Ying-Yung; Hu, Chih-Yung; Lai, Jiann-Shiun

    2016-01-01

    Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%-347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy. PMID:26861297

  16. Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy

    PubMed Central

    Lai, Yan-Da; Wu, Yen-Yu; Tsai, Yi-Jiue; Tsai, Yi-San; Lin, Yu-Ying; Lai, Szu-Liang; Huang, Chao-Yang; Lok, Ying-Yung; Hu, Chih-Yung; Lai, Jiann-Shiun

    2016-01-01

    Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%–347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy. PMID:26861297

  17. Evolution of potent and stable placental-growth-factor-1-targeting CovX-bodies from phage display peptide discovery.

    PubMed

    Bower, Kristen E; Lam, Son N; Oates, Bryan D; Del Rosario, Joselyn R; Corner, Emily; Osothprarop, Trina F; Kinhikar, Arvind G; Hoye, Julie A; Preston, R Ryan; Murphy, Robert E; Campbell, Lioudmila A; Huang, Hanhua; Jimenez, Judith; Cao, Xia; Chen, Gang; Ainekulu, Zemeda W; Datt, Aakash B; Levin, Nancy J; Doppalapudi, Venkata R; Pirie-Shepherd, Steven R; Bradshaw, Curt; Woodnutt, Gary; Lappe, Rodney W

    2011-03-10

    Novel phage-derived peptides are the first reported molecules specifically targeting human placental growth factor 1 (PlGF-1). Phage data enabled peptide modifications that decreased IC(50) values in PlGF-1/VEGFR-1 competition ELISA from 100 to 1 μM. Peptides exhibiting enhanced potency were bioconjugated to the CovX antibody scaffold 1 (CVX-2000), generating bivalent CovX-Bodies with 2 nM K(D) against PlGF-1. In vitro and in vivo peptide cleavage mapping studies enabled the identification of proteolytic hotspots that were subsequently chemically modified. These changes decreased IC(50) to 0.4 nM and increased compound stability from 5% remaining at 6 h after injection to 35% remaining at 24 h with a β phase half-life of 75 h in mice. In cynomolgus monkey, a 78 h β half-life was observed for lead compound 2. The pharmacological properties of 2 are currently being explored. PMID:21280651

  18. Listeria phages

    PubMed Central

    Klumpp, Jochen; Loessner, Martin J

    2013-01-01

    Listeria is an important foodborne pathogen and the causative agent of Listeriosis, a potentially fatal infection. Several hundred Listeria bacteriophages have been described over the past decades, but only few have actually been characterized in some detail, and genome sequences are available for less than twenty of them. We here present an overview of what is currently known about Listeria phage genomics, their role in host evolution and pathogenicity, and their various applications in biotechnology and diagnostics. PMID:24251077

  19. Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor.

    PubMed

    Friedman, M; Nordberg, E; Höidén-Guthenberg, I; Brismar, H; Adams, G P; Nilsson, F Y; Carlsson, J; Ståhl, S

    2007-04-01

    Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 25-50 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents for radionuclide based imaging applications in various carcinomas is discussed. PMID:17452435

  20. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    PubMed

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents. PMID:15518242

  1. Influence of Environmental Factors on Phage–Bacteria Interaction and on the Efficacy and Infectivity of Phage P100

    PubMed Central

    Fister, Susanne; Robben, Christian; Witte, Anna K.; Schoder, Dagmar; Wagner, Martin; Rossmanith, Peter

    2016-01-01

    When using bacteriophages to control food-borne bacteria in food production plants and processed food, it is crucial to consider that environmental conditions influence their stability. These conditions can also affect the physiological state of bacteria and consequently host–virus interaction and the effectiveness of the phage ability to reduce bacteria numbers. In this study we investigated the stability, binding, and replication capability of phage P100 and its efficacy to control Listeria monocytogenes under conditions typically encountered in dairy plants. The influences of SDS, Lutensol AO 7, salt, smear water, and different temperatures were investigated. Results indicate that phage P100 is stable and able to bind to the host under most conditions tested. Replication was dependent upon the growth of L. monocytogenes and efficacy was higher when bacterial growth was reduced by certain environmental conditions. In long-term experiments at different temperatures phages were initially able to reduce bacteria up to seven log10 units after 2 weeks at 4°C. However, thereafter, re-growth and development of phage-resistant L. monocytogenes isolates were encountered. PMID:27516757

  2. Control of Pierce's Disease by Phage

    PubMed Central

    Das, Mayukh; Bhowmick, Tushar Suvra; Ahern, Stephen J.; Young, Ry; Gonzalez, Carlos F.

    2015-01-01

    Pierce’s Disease (PD) of grapevines, caused by Xylella fastidiosa subsp. fastidiosa (Xf), is a limiting factor in the cultivation of grapevines in the US. There are presently no effective control methods to prevent or treat PD. The therapeutic and prophylactic efficacy of a phage cocktail composed of four virulent (lytic) phages was evaluated for control of PD. Xf levels in grapevines were significantly reduced in therapeutically or prophylactically treated grapevines. PD symptoms ceased to progress one week post-therapeutic treatment and symptoms were not observed in prophylactically treated grapevines. Cocktail phage levels increased in grapevines in the presence of the host. No in planta phage-resistant Xf isolates were obtained. Moreover, Xf mutants selected for phage resistance in vitro did not cause PD symptoms. Our results indicate that phages have great potential for biocontrol of PD and other economically important diseases caused by Xylella. PMID:26107261

  3. Control of Pierce's Disease by Phage.

    PubMed

    Das, Mayukh; Bhowmick, Tushar Suvra; Ahern, Stephen J; Young, Ry; Gonzalez, Carlos F

    2015-01-01

    Pierce's Disease (PD) of grapevines, caused by Xylella fastidiosa subsp. fastidiosa (Xf), is a limiting factor in the cultivation of grapevines in the US. There are presently no effective control methods to prevent or treat PD. The therapeutic and prophylactic efficacy of a phage cocktail composed of four virulent (lytic) phages was evaluated for control of PD. Xf levels in grapevines were significantly reduced in therapeutically or prophylactically treated grapevines. PD symptoms ceased to progress one week post-therapeutic treatment and symptoms were not observed in prophylactically treated grapevines. Cocktail phage levels increased in grapevines in the presence of the host. No in planta phage-resistant Xf isolates were obtained. Moreover, Xf mutants selected for phage resistance in vitro did not cause PD symptoms. Our results indicate that phages have great potential for biocontrol of PD and other economically important diseases caused by Xylella. PMID:26107261

  4. Responses to patronizing communication and factors that attenuate those responses.

    PubMed

    Hehman, Jessica A; Bugental, Daphne Blunt

    2015-09-01

    The purpose of this study was to investigate younger (n = 52, ages 18-24) and older (n = 69, ages 61-98) adults' responses to patronizing communication in terms of (a) performance on a cognitive task (Weschler Adult Intelligence Scale-III block design) and (b) physiological responses (i.e., change in cortisol levels), as well as factors that may attenuate those responses. Participants were randomly assigned to receive instructions for the task using either a patronizing or nonpatronizing speech style. Participants also completed a measure of attitudes about aging and the quantity/quality of their intergenerational interaction. Older adults (relative to younger adults) were found to be more reactive to the patronizing speech style in terms of their performance on the task as well as the change in their cortisol levels. Older adults who had more positive attitudes about aging as well as more positive intergenerational interactions were protected from the performance deficits as a result of patronizing speech style. These findings could be used to inform social programs aimed at reducing age-based stigma and improving the life course outcomes of our aging population. PMID:26146886

  5. Hepatic Aryl Hydrocarbon Receptor Attenuates Fibroblast Growth Factor 21 Expression.

    PubMed

    Girer, Nathaniel G; Murray, Iain A; Omiecinski, Curtis J; Perdew, Gary H

    2016-07-15

    The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in many physiological processes. Several studies indicate that AHR is also involved in energy homeostasis. Fibroblast growth factor 21 (FGF21) is an important regulator of the fasting and feeding responses. When administered to various genetic and diet-induced mouse models of obesity, FGF21 can attenuate obesity-associated morbidities. Here, we explore the role of AHR in hepatic Fgf21 expression through the use of a conditional, hepatocyte-targeted AHR knock-out mouse model (Cre(Alb)Ahr(Fx/Fx)). Compared with the congenic parental strain (Ahr(Fx/Fx)), non-fasted Cre(Alb)Ahr(Fx/Fx) mice exhibit a 4-fold increase in hepatic Fgf21 expression, as well as elevated expression of the FGF21-target gene Igfbp1 Furthermore, in vivo agonist activation of AHR reduces hepatic Fgf21 expression during a fast. The Fgf21 promoter contains several putative dioxin response elements (DREs). Using EMSA, we demonstrate that the AHR-ARNT heterodimer binds to a specific DRE that overlaps binding sequences for peroxisome proliferator-activated receptor α (PPARα), carbohydrate response element-binding protein (ChREBP), and cAMP response element-binding protein, hepatocyte specific (CREBH). In addition, we reveal that agonist-activated AHR impairs PPARα-, ChREBP-, and CREBH-mediated promoter activity in Hepa-1 cells. Accordingly, agonist treatment in Hepa-1 cells ablates potent ER stress-driven Fgf21 expression, and pre-treatment with AHR antagonist blocks this effect. Finally, we show that pre-treatment of primary human hepatocytes with AHR agonist diminishes PPARα-, glucose-, and ER stress-driven induction of FGF21 expression, indicating the effect is not mouse-specific. Together, our data show that AHR contributes to hepatic energy homeostasis, partly through the regulation of FGF21 expression and signaling. PMID:27226639

  6. Attenuating properties of hearing protectors in versus time factor.

    PubMed

    Kotarbińska, Ewa

    2003-01-01

    In the noisy environment, workers use hearing protectors to protect their hearing organ against adverse effects of noise. Hearing protectors should be well selected according to the spectrum and level of noise. The selection of hearing protectors is based on the sound attenuation values measured in laboratory conditions. Workers usually use hearing protectors provided by employers as long as there are no signs of any physical damage. The aim of this study was to assess the effect of use and storage as well as of atmospheric conditions on sound attenuation of ear-muffs. Four models of ear-muffs popular in the Polish work environment were tested for two years. They fulfilled the PN-EN 352-1 standard requirements and were granted a certification mark. Fifteen samples of each model were used by workers every working day; 10 samples were exposed to natural atmospheric conditions; and 10 samples were stored in accordance with the manufacture's advice. Temperature and humidity were checked each day. After periods of one and two years, sound attenuation was measured according to PN-EN 24869-1 standard. The observed changes in high-frequency attenuation value H. medium-frequency attenuation value M, low-frequency attenuation value L and single number rating calculated according to PN - EN ISO 4869-2, are presented. The quantitative assessment how far the period of use, storage and exposure to natural atmospheric conditions affect protection achieved by the four popular models of ear-muffs is presented. PMID:12705720

  7. Engineered phages for electronics.

    PubMed

    Cui, Yue

    2016-11-15

    Phages are traditionally widely studied in biology and chemistry. In recent years, engineered phages have attracted significant attentions for functionalization or construction of electronic devices, due to their specific binding, catalytic, nucleating or electronic properties. To apply the engineered phages in electronics, these are a number of interesting questions: how to engineer phages for electronics? How are the engineered phages characterized? How to assemble materials with engineered phages? How are the engineered phages micro or nanopatterned? What are the strategies to construct electronics devices with engineered phages? This review will highlight the early attempts to address these questions and explore the fundamental and practical aspects of engineered phages in electronics, including the approaches for selection or expression of specific peptides on phage coat proteins, characterization of engineered phages in electronics, assembly of electronic materials, patterning of engineered phages, and construction of electronic devices. It provides the methodologies and opens up ex-cit-ing op-por-tu-ni-ties for the development of a variety of new electronic materials and devices based on engineered phages for future applications. PMID:27322923

  8. G5, a Phage Single-Stranded DNA-Binding Protein, Fused with a Nuclear Localization Signal, Attenuates Symptoms and Reduces Begomovirus-Betasatellite Accumulation in Transgenic Plants.

    PubMed

    Rasool, Ghulam; Yousaf, Sumaira; Akram, Afzal; Mansoor, Shahid; Briddon, Rob W; Saeed, Muhammad

    2016-09-01

    Cotton leaf curl disease is caused by several monopartite begomoviruses and is the major threat to cotton production in the Indian subcontinent. The disease has been shown to be associated with four distinct species, including Cotton leaf curl Kokhran virus (CLCuKoV), and a specific betasatellite-Cotton leaf curl Multan betasatellite (CLCuMuB). Transgenic Nicotiana benthamiana plants were produced which constitutively express the Escherichia coli phage M13 encoded, sequence nonspecific single-stranded (ss) DNA-binding protein, G5 alone and fused with the maize opaque-2 nuclear localization signal (NLS), to evaluate resistance against CLCuKoV-CLCuMuB. Transgenic plants expressing only G5 performed poorly exhibiting symptoms of infection and high virus DNA levels upon inoculation with CLCuKoV and CLCuKoV with CLCuMuB. In contrast, plants transformed with G5 fused to the NLS developed mild symptoms and showed a reduction in virus and betasatellite DNA levels in comparison to nontransformed plants. The results show that G5 may be useful in developing broad-spectrum resistance against ssDNA viruses. PMID:27364491

  9. Who went into phage research?

    PubMed Central

    2012-01-01

    A total of 30,000 phage papers, books, or book chapters, published between 1965 and 2010, were analyzed for the ethnic origins of 14,429 first authors. Their names represent 40 linguistic domains or geographic areas and at least 70 languages. British and German names predominate. Results broadly concur with statistics on the frequency of publications by country and show the growing role of Third-World countries in phage research. Irish and Jewish scientists are prominent. Historical and societal factors appear to be very important elements in the advancement of science. PMID:22666657

  10. The peptide growth factor, phytosulfokine, attenuates pattern-triggered immunity.

    PubMed

    Igarashi, Daisuke; Tsuda, Kenichi; Katagiri, Fumiaki

    2012-07-01

    Pattern-triggered immunity (PTI) is triggered by recognition of elicitors called microbe-associated molecular patterns (MAMPs). Although immune responses may provide good protection of plants from pathogen attack, excessive immune responses have negative impacts on plant growth and development. Thus, a good balance between positive and negative effects on the immune signaling network is important for plant fitness. However, little information is known about the molecular mechanisms that are involved in attenuation of PTI. Here, we describe a growth-promoting peptide hormone, phytosulfokine (PSK), as attenuating PTI signaling in Arabidopsis. This research was motivated by the observation that expression of the PSK Receptor 1 (PSKR1) gene was induced by MAMP treatment. Plants homozygous for pskr1 T-DNA insertions showed enhanced defense gene expression and seedling growth inhibition triggered by MAMPs. The pskr1 plants also showed enhanced PTI against the bacterial pathogen Pseudomonas syringae. These results indicate that the PSKR-mediated signaling attenuates immune responses. Tyrosyl protein sulfotransferase (TPST) is an enzyme required for production of the mature sulfated PSK. Like pskr1 mutants, a tpst T-DNA insertion line exhibited enhanced MAMP-triggered seedling growth inhibition, which was suppressed by exogenous application of PSK. Thus, PSK signaling mediated by PSKR1 attenuates PTI but stimulates growth. PMID:22353039

  11. Evaluating risk factors for endemic human Salmonella Enteritidis infections with different phage types in Ontario, Canada using multinomial logistic regression and a case-case study approach

    PubMed Central

    2012-01-01

    Background Identifying risk factors for Salmonella Enteritidis (SE) infections in Ontario will assist public health authorities to design effective control and prevention programs to reduce the burden of SE infections. Our research objective was to identify risk factors for acquiring SE infections with various phage types (PT) in Ontario, Canada. We hypothesized that certain PTs (e.g., PT8 and PT13a) have specific risk factors for infection. Methods Our study included endemic SE cases with various PTs whose isolates were submitted to the Public Health Laboratory-Toronto from January 20th to August 12th, 2011. Cases were interviewed using a standardized questionnaire that included questions pertaining to demographics, travel history, clinical symptoms, contact with animals, and food exposures. A multinomial logistic regression method using the Generalized Linear Latent and Mixed Model procedure and a case-case study design were used to identify risk factors for acquiring SE infections with various PTs in Ontario, Canada. In the multinomial logistic regression model, the outcome variable had three categories representing human infections caused by SE PT8, PT13a, and all other SE PTs (i.e., non-PT8/non-PT13a) as a referent category to which the other two categories were compared. Results In the multivariable model, SE PT8 was positively associated with contact with dogs (OR=2.17, 95% CI 1.01-4.68) and negatively associated with pepper consumption (OR=0.35, 95% CI 0.13-0.94), after adjusting for age categories and gender, and using exposure periods and health regions as random effects to account for clustering. Conclusions Our study findings offer interesting hypotheses about the role of phage type-specific risk factors. Multinomial logistic regression analysis and the case-case study approach are novel methodologies to evaluate associations among SE infections with different PTs and various risk factors. PMID:23057531

  12. Estimating richness from phage metagenomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophages are important drivers of ecosystem functions, yet little is known about the vast majority of phages. Phage metagenomics, or the study of the collective genome of an assemblage of phages, enables the investigation of broad ecological questions in phage communities. One ecological cha...

  13. Phage treatment of human infections

    PubMed Central

    Abedon, Stephen T; Kuhl, Sarah J; Blasdel, Bob G

    2011-01-01

    Phages as bactericidal agents have been employed for 90 years as a means of treating bacterial infections in humans as well as other species, a process known as phage therapy. In this review we explore both the early historical and more modern use of phages to treat human infections. We discuss in particular the little-reviewed French early work, along with the Polish, US, Georgian and Russian historical experiences. We also cover other, more modern examples of phage therapy of humans as differentiated in terms of disease. In addition, we provide discussions of phage safety, other aspects of phage therapy pharmacology, and the idea of phage use as probiotics. PMID:22334863

  14. Attenuation of tumor necrosis factor-induced endothelial cell cytotoxicity and neutrophil chemiluminescence

    SciTech Connect

    Zheng, H.; Crowley, J.J.; Chan, J.C.; Hoffmann, H.; Hatherill, J.R.; Ishizaka, A.; Raffin, T.A. )

    1990-11-01

    Our laboratory has previously shown that the administration of tumor necrosis factor (TNF), a cytokine produced by activated mononuclear cells, to guinea pigs produces a syndrome similar to gram-negative sepsis or ARDS. Pentoxifylline (PTX), a methylxanthine, protects against TNF-induced and sepsis-induced acute lung injury in vivo. We now report on in vitro cellular studies of PMN-mediated cellular injury and its attenuation. We studied TNF-induced bovine pulmonary artery endothelial cell (EC) cytotoxicity both with and without PMN. A 51Cr release assay was used to measure EC damage. Further, we investigated PMN function in response to TNF by measuring chemiluminescence. Agents that attenuate EC damage and PMN activation were evaluated in the above assays. Results revealed that TNF causes EC injury (p less than 0.05) and PMN increase TNF-induced EC injury. Furthermore, PTX, aminophylline (AMPH), caffeine, and forskolin attenuate TNF-induced EC cytotoxicity only in the presence of PMN (p less than 0.05). Of interest, dibutyryl cAMP (DBcAMP) protects EC from TNF-induced injury both with and without PMN. Agents that may increase cAMP levels in PMN (PTX, DBcAMP, forskolin, isobutyl methylxanthine, and terbutaline) significantly attenuate TNF-induced PMN chemiluminescence (p less than 0.05). We conclude that TNF causes EC damage and PMN increase this damage. Furthermore, PTX, AMPH, caffeine, and forskolin can attenuate TNF-induced EC injury in the presence of PMN, whereas DBcAMP attenuates TNF-induced EC injury with and without PMN. In addition, agents that may increase intracellular cAMP levels in PMN can attenuate TNF-induced PMN chemiluminescence. Thus, these agents likely attenuate TNF-induced PMN-mediated EC injury through their inhibitory effects on PMN.

  15. Does Pomegranate intake attenuate cardiovascular risk factors in hemodialysis patients?

    PubMed Central

    2014-01-01

    Background Atherosclerotic cardiovascular disease (CVD) is the most common cause of morbidity and mortality among hemodialysis (HD) patients. It has been attributed, among other causes, to hypertension and dyslipidemia. The aim of the present study was to investigate the effect of a year-long consumption of Pomegranate juice (PJ), on two traditional cardiovascular (CV) risk factors: hypertension and lipid profile, as well as on cardiovascular events. Methods 101 HD patients were randomized to receive 100 cc of PJ (0.7 mM polyphenols) or matching placebo juice, three times a week for one year. The primary endpoints were traditional CV risk factors; blood pressure and lipid profile. Systolic, diastolic and pulse pressure, plasma levels of triglycerides (TG), high density lipoprotein (HDL), low density lipoprotein (LDL) and total cholesterol were monitored quarterly during the study year. Secondary endpoint was incidence of cardiovascular events. Results PJ consumption yielded a significant time response improvement in systolic blood pressure, pulse pressure, triglycerides and HDL level; an improvement that was not observed in the placebo intake group. These beneficial outcomes were more pronounced among patients with hypertension, high level of triglycerides and low levels of HDL. Conclusion Regular PJ consumption by HD patients reduced systolic blood pressure and improved lipid profile. These favorable changes may reduce the accelerated atherosclerosis and high incidence of CVD among HD patients. Trial registration ClinicalTrials.gov registry, Identifier number: NCT00727519 PMID:24593225

  16. The Staphylococci Phages Family: An Overview

    PubMed Central

    Deghorain, Marie; Van Melderen, Laurence

    2012-01-01

    Due to their crucial role in pathogenesis and virulence, phages of Staphylococcus aureus have been extensively studied. Most of them encode and disseminate potent staphylococcal virulence factors. In addition, their movements contribute to the extraordinary versatility and adaptability of this prominent pathogen by improving genome plasticity. In addition to S. aureus, phages from coagulase-negative Staphylococci (CoNS) are gaining increasing interest. Some of these species, such as S. epidermidis, cause nosocomial infections and are therefore problematic for public health. This review provides an overview of the staphylococcal phages family extended to CoNS phages. At the morphological level, all these phages characterized so far belong to the Caudovirales order and are mainly temperate Siphoviridae. At the molecular level, comparative genomics revealed an extensive mosaicism, with genes organized into functional modules that are frequently exchanged between phages. Evolutionary relationships within this family, as well as with other families, have been highlighted. All these aspects are of crucial importance for our understanding of evolution and emergence of pathogens among bacterial species such as Staphylococci. PMID:23342361

  17. Vapor intrusion attenuation factors relative to subslab and source, reconsidered in light of background data

    PubMed Central

    Yao, Yijun; Wu, Yun; Suuberg, Eric M.; Provoost, Jeroen; shen, Rui; Ma, Jianqing; Liu, Jing

    2016-01-01

    The basis upon which recommended attenuation factors for vapor intrusion (VI) have been derived are reconsidered. By making a fitting curve to the plot showing the dependence of observed indoor air concentration (cin) on subslab concentration (css) for residences in EPA database, an analytical equation is obtained to identify the relationship among cin, css and the averaged background level. The new relationship indicates that subslab measurements may serve as a useful guide only if css is above 500 μg / m3. Otherwise, cin is independent of css, with a distribution in good agreements with other studies of background levels. Therefore, employing this screening value (500 μg / m3), new contaminant concentration attenuation factors are proposed for VI, and the values for groundwater-to-indoor and subslab-to-indoor air concentration attenuation factors are 0.004 and 0.02, respectively. The former is applied to examining the reported temporal variations of cin obtained during a long-term monitoring study. The results show that using this new groundwater-to-indoor air concentration attenuation factor also provides a reasonably conservative estimate of cin. PMID:25618001

  18. Phages in nature

    PubMed Central

    Millard, Andrew D; Letarov, Andrey V; Heaphy, Shaun

    2011-01-01

    Bacteriophages or phages are the most abundant organisms in the biosphere and they are a ubiquitous feature of prokaryotic existence. A bacteriophage is a virus which infects a bacterium. Archaea are also infected by viruses, whether these should be referred to as ‘phages’ is debatable, but they are included as such in the scope this article. Phages have been of interest to scientists as tools to understand fundamental molecular biology, as vectors of horizontal gene transfer and drivers of bacterial evolution, as sources of diagnostic and genetic tools and as novel therapeutic agents. Unraveling the biology of phages and their relationship with their hosts is key to understanding microbial systems and their exploitation. In this article we describe the roles of phages in different host systems and show how modeling, microscopy, isolation, genomic and metagenomic based approaches have come together to provide unparalleled insights into these small but vital constituents of the microbial world. PMID:21687533

  19. Phage on the stage

    PubMed Central

    Temple, Louise; Lewis, Lynn

    2015-01-01

    The resurgence of interest in bacteriophages for use in combating antibiotic resistant bacteria is coincident with an urgent call for more effective science education practices, including hands-on learning opportunities. To address this issue, a number of solutions have been proposed, including a large educational experiment, begun in 2007 by the Howard Hughes Medical Institute and currently involving over 85 colleges and universities, which has students discovering unique phages, obtaining images, and purifying phage DNA. A subset of these phage genomes is sequenced and analyzed using bioinformatics tools. Papers describing individual phage discoveries and comparative genomic studies are being published regularly. The vast majority of students in the program are in their first year of college, a critical time in capturing their interest and retaining them as science majors. This viral discovery model is being adopted and modified by a wide variety of educational institutions using a number of different bacterial hosts. In the opinion of the authors, this program and others like it represent a model accessible to virtually any undergraduate setting. And because of these programs, bacteriophage enthusiasts (academics, health professionals, biotechnology companies) can look forward to more well prepared students entering their ranks and should anticipate many more potentially useful phages discovered and characterized. PMID:26442195

  20. Characterization of Five Podoviridae Phages Infecting Citrobacter freundii.

    PubMed

    Hamdi, Sana; Rousseau, Geneviève M; Labrie, Simon J; Kourda, Rim S; Tremblay, Denise M; Moineau, Sylvain; Slama, Karim B

    2016-01-01

    Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus. PMID:27446058

  1. Characterization of Five Podoviridae Phages Infecting Citrobacter freundii

    PubMed Central

    Hamdi, Sana; Rousseau, Geneviève M.; Labrie, Simon J.; Kourda, Rim S.; Tremblay, Denise M.; Moineau, Sylvain; Slama, Karim B.

    2016-01-01

    Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus. PMID:27446058

  2. Phages in the global fruit and vegetable industry.

    PubMed

    Żaczek, M; Weber-Dąbrowska, B; Górski, A

    2015-03-01

    From recent articles, we have learned that phages can constitute a promising alternative in the food industry to eliminate bacterial pathogens from seedlings in greenhouse and field environments, as well as from fresh-cut food products. The fruit and vegetable industry requires quite a different approach than the meat or dairy industry. Several factors can inhibit efficacy of phage treatment such as plant watering or washing ready-to-eat products (water may dilute therapeutic doses), UV irradiation or extensive spreading of phytopathogens by wind, insects or even humans. Spontaneously occurring anomalous weather conditions in different parts of the world also may have an enormous impact on phage persistence in cultivations and on yields. Despite that, some phage preparations are commercially available and, without doubt, are much safer than chemical treatments. Along with increasing worldwide fruit and vegetable consumption, plant diseases and human foodborne illnesses are becoming a serious economic problem, resulting in a focus on optimization of phage treatment. PMID:25410419

  3. Phage display of proteins.

    PubMed

    Kościelska, K; Kiczak, L; Kasztura, M; Wesołowska, O; Otlewski, J

    1998-01-01

    In recent years the phage display approach has become an increasingly popular method in protein research. This method enables the presentation of large peptide and protein libraries on the surface of phage particles from which molecules of desired functional property(ies) can be rapidly selected. The great advantage of this method is a direct linkage between an observed phenotype and encapsulated genotype, which allows fast determination of selected sequences. The phage display approach is a powerful tool in generating highly potent biomolecules, including: search for specific antibodies, determining enzyme specificity, exploring protein-protein and protein-DNA interactions, minimizing proteins, introducing new functions into different protein scaffolds, and searching sequence space of protein folding. In this article many examples are given to illustrate that this technique can be used in different fields of protein science. The phage display has a potential of the natural evolution and its possibilities are far beyond rational prediction. Assuming that we can design the selection agents and conditions we should be able to engineer any desired protein function or feature. PMID:9918498

  4. Determination of the tissue attenuation factor along two major axes of a high dose rate (HDR) 192Ir source.

    PubMed

    Cho, S H; Muller-Runkel, R; Hanson, W F

    1999-08-01

    Quantitative information on photon scattering around brachytherapy sources is needed to develop dose calculation formalisms capable of predicting dosimetric parameters with minimal empiricism. Photon absorption and scatter around brachytherapy sources can be characterized using the tissue attenuation factor, defined as the ratio of dose in water to water kerma in free space. In this study, the tissue attenuation factor along two major axes of a high dose rate (HDR) 192Ir source was determined by TLD measurements and MCNP Monte Carlo calculations. A calculational method is also suggested to derive the tissue attenuation factor along the longitudinal source axis from the factor along the transverse axis, using published anisotropy data as input. TLD and Monte Carlo results agreed with each other for both source axes within the statistical uncertainty (approximately +/- 5%) of Monte Carlo calculations. Comparison with published data, available only for the transverse source axis, also showed good agreement within +/- 5%. The shape and magnitude of the tissue attenuation factor are found to be remarkably different between the two axes. The tissue attenuation factor reaches a maximum value of about 1.4 at 8 cm from the source along the longitudinal source axis, while a maximum value of about 1.04 occurs at 3-4 cm from the source along the transverse axis. The calculated tissue attenuation factor along the longitudinal source axis generally reproduced the TLD and Monte Carlo results within +/- 5% at most radial distances. PMID:10501048

  5. Angiotensin peptides attenuate platelet-activating factor-induced inflammatory activity in rats.

    PubMed

    Sato, Akira; Yokoyama, Izumi; Ebina, Keiichi

    2015-11-01

    Angiotensin (Ang)--a peptide that is part of the renin-angiotensin system-induces vasoconstriction and a subsequent increase in blood pressure; Ang peptides, especially AngII, can also act as potent pro-inflammatory mediators. Platelet-activating factor (PAF) is a potent phospholipid mediator that is implicated in many inflammatory diseases. In this study, we investigated the effects of Ang peptides (AngII, AngIII, and AngIV) on PAF-induced inflammatory activity. In experiments using a rat hind-paw oedema model, AngII markedly and dose-dependently attenuated the paw oedema induced by PAF. The inhibitory effects of AngIII and AngIV on PAF-induced paw oedema were lower than that of AngII. Two Ang receptors, the AT1 and AT2 receptors, did not affect the AngII-mediated attenuation of PAF-induced paw oedema. Moreover, intrinsic tyrosine fluorescence studies demonstrated that AngII, AngIII, and AngIV interact with PAF, and that their affinities were closely correlated with their inhibitory effects on PAF-induced rat paw oedema. Also, AngII interacted with metabolite/precursor of PAF (lyso-PAF), and an oxidized phospholipid, 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), which bears a marked structural resemblance to PAF. Furthermore, POVPC dose-dependently inhibited AngII-mediated attenuation of PAF-induced paw oedema. These results suggest that Ang peptides can attenuate PAF-induced inflammatory activity through binding to PAF and lyso-PAF in rats. Therefore, Ang peptides may be closely involved in the regulation of many inflammatory diseases caused by PAF. PMID:26348270

  6. Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood.

    PubMed

    Majewska, Joanna; Beta, Weronika; Lecion, Dorota; Hodyra-Stefaniak, Katarzyna; Kłopot, Anna; Kaźmierczak, Zuzanna; Miernikiewicz, Paulina; Piotrowicz, Agnieszka; Ciekot, Jarosław; Owczarek, Barbara; Kopciuch, Agnieszka; Wojtyna, Karolina; Harhala, Marek; Mąkosa, Mateusz; Dąbrowska, Krystyna

    2015-08-01

    A specific humoral response to bacteriophages may follow phage application for medical purposes, and it may further determine the success or failure of the approach itself. We present a long-term study of antibody induction in mice by T4 phage applied per os: 100 days of phage treatment followed by 112 days without the phage, and subsequent second application of phage up to day 240. Serum and gut antibodies (IgM, IgG, secretory IgA) were analyzed in relation to microbiological status of the animals. T4 phage applied orally induced anti-phage antibodies when the exposure was long enough (IgG day 36, IgA day 79); the effect was related to high dosage. Termination of phage treatment resulted in a decrease of IgA again to insignificant levels. Second administration of phage induces secretory IgA sooner than that induced by the first administrations. Increased IgA level antagonized gut transit of active phage. Phage resistant E. coli dominated gut flora very late, on day 92. Thus, the immunological response emerges as a major factor determining phage survival in the gut. Phage proteins Hoc and gp12 were identified as highly immunogenic. A low response to exemplary foreign antigens (from Ebola virus) presented on Hoc was observed, which suggests that phage platforms can be used in oral vaccine design. PMID:26308042

  7. Phage Neutralization by Sera of Patients Receiving Phage Therapy

    PubMed Central

    Żaczek, Maciej; Weber-Dąbrowska, Beata; Międzybrodzki, Ryszard; Kłak, Marlena; Fortuna, Wojciech; Letkiewicz, Sławomir; Rogóż, Paweł; Szufnarowski, Krzysztof; Jończyk-Matysiak, Ewa; Owczarek, Barbara; Górski, Andrzej

    2014-01-01

    Abstract The aim of our investigation was to verify whether phage therapy (PT) can induce antiphage antibodies. The antiphage activity was determined in sera from 122 patients from the Phage Therapy Unit in Wrocław with bacterial infections before and during PT, and in sera from 30 healthy volunteers using a neutralization test. Furthermore, levels of antiphage antibodies were investigated in sera of 19 patients receiving staphylococcal phages and sera of 20 healthy volunteers using enzyme-linked immunosorbent assay. The phages were administered orally, locally, orally/locally, intrarectally, or orally/intrarectally. The rate of phage inactivation (K) estimated the level of phages' neutralization by human sera. Low K rates were found in sera of healthy volunteers (K≤1.73). Low K rates were detected before PT (K≤1.64). High antiphage activity of sera K>18 was observed in 12.3% of examined patients (n=15) treated with phages locally (n=13) or locally/orally (n=2) from 15 to 60 days of PT. High K rates were found in patients treated with some Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis phages. Low K rates were observed during PT in sera of patients using phages orally (K≤1.04). Increased inactivation of phages by sera of patients receiving PT decreased after therapy. These results suggest that the antiphage activity in patients' sera depends on the route of phage administration and phage type. The induction of antiphage activity of sera during or after PT does not exclude a favorable result of PT. PMID:24893003

  8. Marine phages as excellent tracers for reactive colloidal transport in porous media

    NASA Astrophysics Data System (ADS)

    Ghanem, Nawras; Chatzinotas, Antonis; Harms, Hauke; Wick, Lukas Y.

    2016-04-01

    Question: Here we evaluate marine phages as specific markers of hydrological flow and reactive transport of colloidal particles in the Earth's critical zone (CZ). Marine phages and their bacterial hosts are naturally absent in the CZ, and can be detected with extremely high sensitivity. In the framework of the DFG Collaborative Research Center AquaDiva, we asked the following questions: (1) Are marine phages useful specific markers of hydrological flow and reactive transport in porous media? and (2) Which phage properties are relevant drivers for the transport of marine phages in porous media? Methods: Seven marine phages from different families (as well two commonly used terrestrial phages) were selected based on their morphology, size and physico-chemical surface properties (surface charge and hydrophobicity). Phage properties were assessed by electron microscopy, dynamic light scattering and water contact angle analysis (CA). Sand-filled laboratory percolation columns were used to study transport. The breakthrough curves of the phages were analyzed using the clean bed filtration theory and the XDLVO theory of colloid stability, respectively. Phages were quantified by a modified high- throughput plaque assay and a culture-independent particle counting method approach. Results: Our data show that most marine tested phages exhibited highly variable transport rates and deposition efficiency, yet generally high colloidal stability and viability. We find that size, morphology and hydrophobicity are key factors shaping the transport efficiency of phages. Differing deposition efficiencies of the phages were also supported by calculated XDLVO interaction energy profile. Conclusion: Marine phages have a high potential for the use as sensitive tracers in terrestrial habitats with their surface properties playing a crucial role for their transport. Marine phages however, exhibit differences in their deposition efficiency depending on their morphology, hydrophobicity and

  9. The first EGF domain of coagulation factor IX attenuates cell adhesion and induces apoptosis.

    PubMed

    Ishikawa, Tomomi; Kitano, Hisataka; Mamiya, Atsushi; Kokubun, Shinichiro; Hidai, Chiaki

    2016-07-01

    Coagulation factor IX (FIX) is an essential plasma protein for blood coagulation. The first epidermal growth factor (EGF) motif of FIX (EGF-F9) has been reported to attenuate cell adhesion to the extracellular matrix (ECM). The purpose of the present study was to determine the effects of this motif on cell adhesion and apoptosis. Treatment with a recombinant EGF-F9 attenuated cell adhesion to the ECM within 10 min. De-adhesion assays with native FIX recombinant FIX deletion mutant proteins suggested that the de-adhesion activity of EGF-F9 requires the same process of FIX activation as that which occurs for coagulation activity. The recombinant EGF-F9 increased lactate dehydrogenase (LDH) activity release into the medium and increased the number of cells stained with annexin V and activated caspase-3, by 8.8- and 2.7-fold respectively, indicating that EGF-F9 induced apoptosis. Activated caspase-3 increased very rapidly after only 5 min of administration of recombinant EGF-F9. Treatment with EGF-F9 increased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), but not that of phosphorylated MAPK 44/42 or c-Jun N-terminal kinase (JNK). Inhibitors of caspase-3 suppressed the release of LDH. Caspase-3 inhibitors also suppressed the attenuation of cell adhesion and phosphorylation of p38 MAPK by EGF-F9. Our data indicated that EGF-F9 activated signals for apoptosis and induced de-adhesion in a caspase-3 dependent manner. PMID:27129300

  10. Two different evolutionary lines of filamentous phages in Ralstonia solanacearum: their effects on bacterial virulence

    PubMed Central

    Askora, Ahmed; Yamada, Takashi

    2015-01-01

    The integration and excision of various filamentous phage genomes into and out of their host chromosomes occurs by site-specific recombination. The mechanisms proposed for these events include reactions mediated by phage-encoded recombinases and host recombination systems. Site-specific integration of filamentous phages plays a vital role in a variety of biological functions of the host, such as phase variation of certain pathogenic bacterial virulence factors. The importance of these filamentous phages in bacterial evolution is rapidly increasing with the discovery of new phages that are involved in pathogenicity. Studies of the diversity of two different filamentous phages infecting the phytopathogen Ralstonia solanacearum provide us with novel insights into the dynamics of phage genomes, biological roles of prophages, and the regulation and importance of phage–host interactions. PMID:26150828

  11. Why do phage play dice?

    PubMed

    Avlund, Mikkel; Dodd, Ian B; Semsey, Szabolcs; Sneppen, Kim; Krishna, Sandeep

    2009-11-01

    Phage lambda is among the simplest organisms that make a developmental decision. An infected bacterium goes either into the lytic state, where the phage particles rapidly replicate and eventually lyse the cell, or into a lysogenic state, where the phage goes dormant and replicates along with the cell. Experimental observations by P. Kourilsky are consistent with a single phage infection deterministically choosing lysis and double infection resulting in a stochastic choice. We argue that the phage are playing a "game" of minimizing the chance of extinction and that the shift from determinism to stochasticity is due to a shift from a single-player to a multiplayer game. Crucial to the argument is the clonal identity of the phage. PMID:19740995

  12. CLAUSA Is a MYB Transcription Factor That Promotes Leaf Differentiation by Attenuating Cytokinin Signaling.

    PubMed

    Bar, Maya; Israeli, Alon; Levy, Matan; Ben Gera, Hadas; Jiménez-Gómez, José M; Kouril, Stepan; Tarkowski, Petr; Ori, Naomi

    2016-07-01

    Leaf morphogenesis and differentiation are highly flexible processes, resulting in a large diversity of leaf forms. The development of compound leaves involves an extended morphogenesis stage compared with that of simple leaves, and the tomato (Solanum lycopersicum) mutant clausa (clau) exposes a potential for extended morphogenesis in tomato leaves. Here, we report that the CLAU gene encodes a MYB transcription factor that has evolved a unique role in compound-leaf species to promote an exit from the morphogenetic phase of tomato leaf development. We show that CLAU attenuates cytokinin signaling, and that clau plants have increased cytokinin sensitivity. The results suggest that flexible leaf patterning involves a coordinated interplay between transcription factors and hormones. PMID:27385816

  13. Sinorhizobium meliloti Phage ΦM9 Defines a New Group of T4 Superfamily Phages with Unusual Genomic Features but a Common T=16 Capsid

    PubMed Central

    Johnson, Matthew C.; Tatum, Kelsey B.; Lynn, Jason S.; Brewer, Tess E.; Lu, Stephen; Washburn, Brian K.

    2015-01-01

    , contractile tail through which the DNA is delivered to host cells. This phylogenetic and structural study of S. meliloti-infecting T4 superfamily phage ΦM9 provides new insight into the diversity of this family. The comparison of structure-related genes in both ΦM9 and S. meliloti-infecting T4 superfamily phage ΦM12, which comes from a completely different lineage of these phages, allows the identification of host infection-related factors. PMID:26311868

  14. Ultrasound-targeted antisense oligonucleotide attenuates ischemia/reperfusion-induced myocardial tumor necrosis factor-alpha.

    PubMed

    Erikson, John M; Freeman, Gregory L; Chandrasekar, Bysani

    2003-01-01

    Ultrasound contrast agents are now emerging as effective vehicles for delivering therapeutic agents to target tissues. In the present study, we used ultrasound-targeted, contrast-bound antisense oligonucleotides to inhibit the expression of tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine with negative inotropic effects. We compared the efficacy of left ventricular vs. intravenous administration and determined the optimal time for delivery. WKY rats were treated with perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microspheres incubated with 100 microg of antisense oligonucleotide directed against TNF-alpha. Contrast was infused into either the superior vena cava or the left ventricular cavity along with simultaneous application of ultrasound. Twenty-four hours later, the animals underwent 15 min of ischemia and 2 h reperfusion. Control animals underwent sham operation only, ischemia/reperfusion only, or received PESDA only. A second group received treatment just prior to, or immediately after the onset of ischemia. At the end of the experimental period, hearts were removed and analyzed for TNF-alpha by northern and western blotting. While no TNF-alpha expression was detected in sham-operated animals, robust expression of TNF-alpha mRNA and protein was seen in controls treated with ultrasound and PESDA alone. In contrast, intravenous or left ventricular administration of antisense oligonucleotides significantly inhibited ischemia/reperfusion-induced TNF-alpha expression. Direct delivery into the left ventricular cavity was more effective than intravenous administration, and delivery just prior to ischemia was most effective in attenuating TNF-alpha expression. Furthermore, attenuation of TNF-alpha expression also significantly inhibited other post-ischemic inflammatory mediators including IL-1beta and intercellular adhesion molecule-1 (ICAM-1). Thus, ultrasound-targeted antisense oligonucleotides can effectively attenuate post

  15. Attenuation of internal organ damages by exogenously administered epidermal growth factor (EGF) in burned rodents.

    PubMed

    Berlanga, Jorge; Lodos, Jorge; López-Saura, Pedro

    2002-08-01

    Major burns are associated with multiple internal organ damages, including necrosis of the gastrointestinal mucosa. Failure of the intestinal barrier is a serious complication in burned patients. Epidermal growth factor (EGF) is a mitogenic polypeptide that stimulates wound repair and affords protection to the gastric mucosa. We examined whether a single systemic intervention with EGF prevents organ systems damages, following full-thickness scalds (25-30%) in rodents. Animals were randomly assigned to receive an intraperitoneal injection of EGF (30 microg/kg in mice, 10 microg/kg in rats) or saline solution, 30 min prior thermal injury in mice or after the cutaneous injury in rats. General clinical condition and mortality during 24h were recorded. Animals were autopsied and histopathological and histomorphometric studies were conducted. Mice treated with EGF exhibited a milder clinical evolution and acute lethality was significantly reduced as compared to saline counterparts (P<0.01). Histopathological and morphometric analysis showed that EGF significantly reduced intestinal necrosis and contributed to preserve jejunoileal architecture in mice (P<0.05) and rats (P<0.01). The onset of renal hemorrhagic foci was significantly reduced in EGF-treated groups (P<0.01). Lung damages appeared attenuated in EGF-treated animals. These data indicate the salutary effects of EGF by attenuating internal complications associated to thermal injuries. Further studies are warranted to fully elucidate the usefulness of this therapy. PMID:12163282

  16. MTMR4 attenuates transforming growth factor beta (TGFbeta) signaling by dephosphorylating R-Smads in endosomes.

    PubMed

    Yu, Junjing; Pan, Lei; Qin, Xincheng; Chen, Hua; Xu, Youli; Chen, Yeguang; Tang, Hong

    2010-03-12

    Homeostasis of Smad phosphorylation at its C-terminal SXS motif is essential for transforming growth factor beta (TGFbeta) signaling. Whereas it is known that TGFbeta signaling can be terminated by phosphatases, which dephosphorylate R-Smads in the nucleus, it is unclear whether there are any cytoplasmic phosphatase(s) that can attenuate R-Smad phosphorylation and nuclear translocation. Here we demonstrate that myotubularin-related protein 4 (MTMR4), a FYVE domain-containing dual-specificity protein phosphatase (DSP), attenuates TGFbeta signaling by reducing the phosphorylation level of R-Smads in early endosomes. Co-immunoprecipitation experiments showed that endogenous MTMR4 interacts with phosphorylated R-Smads, and that this interaction is correlated with dephosphorylation of R-Smads. Further analysis showed that overexpression of MTMR4 resulted in the sequestration of activated Smad3 in the early endosomes, thus reducing its nuclear translocation. However, both point mutations at the conserved catalytic site of the phosphatase (MTMR4-C407S) and small interference RNA of endogenous Mtmr4 expression led to sustained Smad3 activation. This work therefore suggests that MTMR4 plays an important role in preventing the overactivation of TGFbeta signaling by dephosphorylating activated R-Smads that have been trafficked to early endosomes. PMID:20061380

  17. Clostridium difficile phages: still difficult?

    PubMed Central

    Hargreaves, Katherine R.; Clokie, Martha R. J.

    2014-01-01

    Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however, developing suitable phages is challenging. In this review we summarize the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics. Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage–host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution. No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using “whole-phages” are such that they remain a major research focus. Whilst we don’t envisage working with C. difficile phages will be problem-free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen. PMID:24808893

  18. Examination of the influence of environmental factors in contaminant vapor concentration attenuation factor with U.S. EPA’s vapor intrusion database

    PubMed Central

    Yao, Yijun; Shen, Rui; Pennell, Kelly G.; Suuberg, Eric M.

    2013-01-01

    Those charged with the responsibility of estimating the risk posed by vapor intrusion (VI) processes have often looked to information contained in the U.S. Environmental Protection Agency (EPA)’s VI database for insight. Indoor air concentration attenuation factors have always been a key focus of this database, but the roles of different environmental factors in these attenuation processes are still unclear. This study aims to examine the influences of these factors in the context of the information in the VI database. The database shows that the attenuation factors vary over many orders of magnitude, and that no simple statistical fluctuation around any typical mean value exists. Thus far, no simple explanation of this phenomenon has been presented. This paper examines various possible contributing factors to the enormous range of observed values, looking at which ones can plausibly contribute to explaining them. PMID:23252837

  19. Phage and Yeast Display.

    PubMed

    Sheehan, Jared; Marasco, Wayne A

    2015-02-01

    Despite the availability of antimicrobial drugs, the continued development of microbial resistance--established through escape mutations and the emergence of resistant strains--limits their clinical utility. The discovery of novel, therapeutic, monoclonal antibodies (mAbs) offers viable clinical alternatives in the treatment and prophylaxis of infectious diseases. Human mAb-based therapies are typically nontoxic in patients and demonstrate high specificity for the intended microbial target. This specificity prevents negative impacts on the patient microbiome and avoids driving the resistance of nontarget species. The in vitro selection of human antibody fragment libraries displayed on phage or yeast surfaces represents a group of well-established technologies capable of generating human mAbs. The advantage of these forms of microbial display is the large repertoire of human antibody fragments present during a single selection campaign. Furthermore, the in vitro selection environments of microbial surface display allow for the rapid isolation of antibodies--and their encoding genes--against infectious pathogens and their toxins that are impractical within in vivo systems, such as murine hybridomas. This article focuses on the technologies of phage display and yeast display, as these strategies relate to the discovery of human mAbs for the treatment and vaccine development of infectious diseases. PMID:26104550

  20. A revision factor to the Cutshall self-attenuation correction in (210)Pb gamma-spectrometry measurements.

    PubMed

    Jodłowski, Paweł

    2016-03-01

    The Cutshall transmission method of determination of self-attenuation correction in (210)Pb measurements by gamma-spectrometry gives the results burdened with errors of up to 10%. The author proposes introducing into the Cutshall correction Cs,Cuts an additional revision factor CCs,Cuts to eliminate errors. The proposed formula of the revision factor describes the CCs,Cuts value depending on the experimentally obtained Cs,Cuts correction. Formula holds true in wide ranges of the measurement geometries and linear attenuation coefficients of both the standard and the sample. PMID:26702546

  1. Estimation of patient attenuation factor for iodine-131 based on direct dose rate measurements from radioiodine therapy patients.

    PubMed

    Soliman, Khaled; Alenezi, Ahmed

    2015-02-01

    The aim of the study was to measure the actual dose at 1 m from the patients per unit activity with the aim of providing a more accurate prediction of the dose levels around radioiodine patients in the hospital, as well as to compare our results with the literature. In this work the demonstration of a patient body tissue attenuation factor is verified by comparing the dose rates measured from the patients with those measured from the unshielded radioiodine capsules immediately after administration of the radioactivity. The normalized dose rate per unit activity is therefore proposed as an operational quantity that can be used to predict exposure rates to staff and patients' relatives. The average dose rate measured from our patient per unit activity was 38.4±11.8 μSv/h/GBq. The calculated attenuation correction factor based on our measurements was 0.55±0.17. The calculated dose rate from a radioiodine therapy patient should normally include a factor accounting for patient body tissue attenuation and scatter. The attenuation factor is currently neglected and not applied in operational radiation protection. Realistic estimation of radiation dose levels from radioiodine therapy patients when properly performed will reduce the operational cost and optimize institutional radiation protection practice. It is recommended to include patient attenuation factors in risk assessment exercises - in particular, when accurate estimates of total effective doses to exposed individuals are required when direct measurements are not possible. The information provided about patient attenuation might benefit radiation protection specialists and regulators. PMID:25279710

  2. Inactivation of Escherichia coli phage by pulsed electric field treatment and analysis of inactivation mechanism

    NASA Astrophysics Data System (ADS)

    Tanino, Takanori; Yoshida, Tomoki; Sakai, Kazuki; Ohshima, Takayuki

    2013-03-01

    Inactivation of bacteriophage by pulsed electric field (PEF) treatment, one of the effective procedures for bacteria nonthermal inactivation, was studied. Model phage particles Escherichia coli bacteriophages M13mp18 and λ phage, were successfully inactivated by PEF treatment. The survival ratios of both bacteriophages decreased depending on the PEF treatment time when applied peak voltage was 5 or 7 kV, and the survival ratios after 12 min PEF treatment were 10-4 - 10-5. Electrophoresis analyses of biological molecules of inactivated λ phage detected no degradation of total protein and genomic DNA. These results suggested that the factor of phage inactivation by PEF treatment was not based on the degradation of protein or DNA, but on the destruction of phage particle structure. Sensitivity of E. coli phage to PEF treatment was compared with that of E. coli cell. Phage and MV1184 cell were treated with same condition PEF at 5 kV, respectively. After 12 min treatment, the survival ration of λ phage and MV1184 were 4.0 × 10-5 and 1.7 × 10-3, respectively. The survival ratio of phage was lower than that of MV1184. E. coli cell is more tolerant to inactivation with PEF treatment than coli phage.

  3. Granulocyte colony stimulating factor attenuates inflammation in a mouse model of amyotrophic lateral sclerosis

    PubMed Central

    2011-01-01

    Background Granulocyte colony stimulating factor (GCSF) is protective in animal models of various neurodegenerative diseases. We investigated whether pegfilgrastim, GCSF with sustained action, is protective in a mouse model of amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease with manifestations of upper and lower motoneuron death and muscle atrophy accompanied by inflammation in the CNS and periphery. Methods Human mutant G93A superoxide dismutase (SOD1) ALS mice were treated with pegfilgrastim starting at the presymptomatic stage and continued until the end stage. After long-term pegfilgrastim treatment, the inflammation status was defined in the spinal cord and peripheral tissues including hematopoietic organs and muscle. The effect of GCSF on spinal cord neuron survival and microglia, bone marrow and spleen monocyte activation was assessed in vitro. Results Long-term pegfilgrastim treatment prolonged mutant SOD1 mice survival and attenuated both astro- and microgliosis in the spinal cord. Pegfilgrastim in SOD1 mice modulated the inflammatory cell populations in the bone marrow and spleen and reduced the production of pro-inflammatory cytokine in monocytes and microglia. The mobilization of hematopoietic stem cells into the circulation was restored back to basal level after long-term pegfilgrastim treatment in SOD1 mice while the storage of Ly6C expressing monocytes in the bone marrow and spleen remained elevated. After pegfilgrastim treatment, an increased proportion of these cells in the degenerative muscle was detected at the end stage of ALS. Conclusions GCSF attenuated inflammation in the CNS and the periphery in a mouse model of ALS and thereby delayed the progression of the disease. This mechanism of action targeting inflammation provides a new perspective of the usage of GCSF in the treatment of ALS. PMID:21711557

  4. Platelet-activating factor attenuation of long-term potentiation in rat hippocampal slices via protein tyrosine kinase signaling.

    PubMed

    Reiner, Benjamin; Wang, Wenwei; Liu, Jianuo; Xiong, Huangui

    2016-02-26

    It is well established that HIV-1-infected mononuclear phagocytes release platelet activating factor (PAF) and elevated levels of PAF have been detected in blood and in the cerebrospinal fluid (CSF) of acquired immunodeficiency syndrome (AIDS) patients with HIV-associated neurocognitive disorders (HAND). It is our hypothesis that the elevated levels of PAF alter long-term potentiation (LTP) in the hippocampus, leading to neurocognitive dysfunction. To test this hypothesis, we studied the effects of PAF on LTP in the CA1 region of rat hippocampal slices. Our results showed incubation of hippocampal slices with PAF attenuated LTP. The PAF-mediated attenuation was blocked by ginkgolide B, a PAF receptor antagonist, suggesting PAF attenuation of LTP via PAF receptors. Application of lyso-PAF, an inactive PAF analog, had no apparent effect on LTP. Further investigation revealed an involvement of tyrosine kinase in PAF attenuation of LTP, which was demonstrated by lavendustin A (a specific protein tyrosine kinase inhibitor) blockage of PAF attenuation of LTP. As LTP is widely considered as the cellular and synaptic basis for learning and memory, the attenuation of LTP by PAF may contribute at least in part to the HAND pathogenesis. PMID:26808643

  5. Phage therapy of pulmonary infections

    PubMed Central

    Abedon, Stephen T

    2015-01-01

    It is generally agreed that a bacteriophage-associated phenomenon was first unambiguously observed one-hundred years ago with the findings of Twort in 1915. This was independently followed by complementary observations by d'Hérelle in 1917. D'Hérelle's appreciation of the bacteriophage phenomenon appears to have directly led to the development of phages as antibacterial agents within a variety of contexts, including medical and agricultural. Phage use to combat nuisance bacteria appears to be especially useful where targets are sufficiently problematic, suitably bactericidal phages exist, and alternative approaches are lacking in effectiveness, availability, safety, or cost effectiveness, etc. Phage development as antibacterial agents has been strongest particularly when antibiotics have been less available or useful, e.g., such as in the treatment of chronic infections by antibiotic-resistant bacteria. One relatively under-explored or at least not highly reported use of phages as therapeutic agents has been to combat bacterial infections of the lungs and associated tissues. These infections are diverse in terms of their etiologies, manifestations, and also in terms of potential strategies of phage delivery. Here I review the literature considering the phage therapy of pulmonary and pulmonary-related infections, with emphasis on reports of clinical treatment along with experimental treatment of pulmonary infections using animal models. PMID:26442188

  6. High Stability of Stx2 Phage in Food and under Food-Processing Conditions ▿

    PubMed Central

    Rode, Tone Mari; Axelsson, Lars; Granum, Per Einar; Heir, Even; Holck, Askild; L'Abée-Lund, Trine M.

    2011-01-01

    Bacteriophages (phages) carrying Shiga toxin genes constitute a major virulence attribute in enterohemorrhagic Escherichia coli (EHEC). Several EHEC outbreaks have been linked to food. The survival of such strains in different foods has received much attention, while the fate of the mobile Shiga toxin-converting phages (Stx phages) has been less studied. We have investigated the stability of an Stx phage in several food products and examined how storage, food processing, and disinfection influence the infectivity of phage particles. The study involved a recombinant Stx phage (Δstx::cat) of an E. coli O103:H25 strain from a Norwegian outbreak in 2006. Temperature, matrix, and time were factors of major importance for the stability of phage particles. Phages stored at cooling temperatures (4°C) showed a dramatic reduction in stability compared to those stored at room temperature. The importance of the matrix was evident at higher temperatures (60°C). Phages in ground beef were below the detection level when heated to 60°C for more than 10 min, while phages in broth exposed to the same heating conditions showed a 5-log-higher stability. The phages tolerated desiccation poorly but were infective for a substantial period of time in solutions. Under moist conditions, they also had a high ability to tolerate exposure to several disinfectants. In a dry-fermented sausage model, phages were shown to infect E. coli in situ. The results show that Stx phage particles can maintain their infectivity in foods and under food-processing conditions. PMID:21685156

  7. Renin-angiotensin blockade resets podocyte epigenome through Kruppel-like Factor 4 and attenuates proteinuria.

    PubMed

    Hayashi, Kaori; Sasamura, Hiroyuki; Nakamura, Mari; Sakamaki, Yusuke; Azegami, Tatsuhiko; Oguchi, Hideyo; Tokuyama, Hirobumi; Wakino, Shu; Hayashi, Koichi; Itoh, Hiroshi

    2015-10-01

    Proteinuria is a central component of chronic kidney disease and an independent risk factor for cardiovascular disease. Kidney podocytes have an essential role as a filtration barrier against proteinuria. Kruppel-like Factor 4 (KLF4) is expressed in podocytes and decreased in glomerular diseases leading to methylation of the nephrin promoter, decreased nephrin expression and proteinuria. Treatment with an angiotensin receptor blocker (ARB) reduced methylation of the nephrin promoter in murine glomeruli of an adriamycin nephropathy model with recovery of KLF4 expression and a decrease in albuminuria. In podocyte-specific KLF4 knockout mice, the effect of ARB on albuminuria and the nephrin promoter methylation was attenuated. In cultured human podocytes, angiotensin II reduced KLF4 expression and caused methylation of the nephrin promoter with decreased nephrin expression. In patients, nephrin promoter methylation was increased in proteinuric kidney diseases with decreased KLF4 and nephrin expression. KLF4 expression in ARB-treated patients was higher in patients with than without ARB treatment. Thus, angiotensin II can modulate epigenetic regulation in podocytes and ARB inhibits these actions in part via KLF4 in proteinuric kidney diseases. This study provides a new concept that renin-angiotensin system blockade can exert therapeutic effects through epigenetic modulation of the kidney gene expression. PMID:26108068

  8. [Biology of two lysogenic phages from Bacillus thuringiensis MZ1].

    PubMed

    Liao, Wei; Sun, Fan; Song, Shao-yun; Shi, Wei; Pang, Yi

    2007-02-01

    A Bacillus thuringiensis (Bt) fermentative strain MZ1 (subsp. kurstaki) , from a company in Meixian County of Guangdong Province, produce toxins during sporulation and are extensively used in the field to control pest insects (Lepidoptera) in China. But some unknown or random factors that inhibited or stopped B. t growth in the fermentation can be regarded as reflecting the exist of lysogenic phage. Therefore, strain MZI and its lysogenic phages were studied in this paper. With indicator strain ZK1, two kind of phage plaques, one with about 3mm diameter and the other with about 1mm diameter, can be observed after strain MZ1 cultured in plates or flasks was induced by mitomycin C. Then, two lysogenic phages, namely MZTP01 and MZTP02, were isolated and characterized in biology. They belonged to family Siphoviridae, which had icosahedral heads (MZTP01 :82nm x 85nm; MZTP02: 75nm x 55nm) and long tails (MZTP01: 220nm x 18nm; MZTP02: 183nm x 12nm) without flexibility. Host range examination showed that six and seven (including indicator strain ZKl) out of 113 B. t strains saved in our laboratory were sensitive to MZTP01 and MZTP02, respectively. MZTP01 was more stable than MZTP02 against pH value, ultraviolet and heat treatment, but contrary against organic solvents. For MZTP01 and MZTP02, K values in the neutralization reactions were 45 and 326, respectively. Both phages had no relationship in their antigenicity. Burst size of phage MZTP02 was 175, two times more than that of MZTP01. Latent time of MZTP02 was 40min, one times shorter than that of MZTP01. It was suggested that both phages DNA be linear dsDNA through the typical absorption curves, reaction with diphenylamine, DNase sensitivity and acridine orange staining. And this was in good accord with the previous findings that all tailed phages being dsDNA moleculars. The genomic DNA and their restriction maps showed that both molecular weights should be between 9.4 - 23kb. Both phage genomic DNAs were digested by

  9. Random Transposon Mutagenesis for Cell-Envelope Resistant to Phage Infection.

    PubMed

    Reyes-Cortés, Ruth; Arguijo-Hernández, Emma S; Carballo-Ontiveros, Marco A; Martínez-Peñafiel, Eva; Kameyama, Luis

    2016-01-01

    In order to identify host components involved in the infective process of bacteriophages, we developed a wide-range strategy to obtain cell envelope mutants, using Escherichia coli W3110 and its specific phage mEp213. The strategy consisted in four steps: (1) random mutagenesis using transposon miniTn10Km(r); (2) selection of phage-resistant mutants by replica-plating; (3) electroporation of the phage-resistant mutants with mEp213 genome, followed by selection of those allowing phage development; and (4) sequencing of the transposon-disrupted genes. This strategy allowed us to distinguish the host factors related to phage development or multiplication within the cell, from those involved in phage infection at the level of the cell envelope. PMID:27311665

  10. The phage-host arms race: Shaping the evolution of microbes

    SciTech Connect

    Stern, Adi; Sorek, Rotem

    2010-10-26

    Bacteria, the most abundant organisms on the planet, are outnumbered by a factor of 10 to 1 by phages that infect them. Faced with the rapid evolution and turnover of phage particles, bacteria have evolved various mechanisms to evade phage infection and killing, leading to an evolutionary arms race. The extensive co-evolution of both phage and host has resulted in considerable diversity on the part of both bacterial and phage defensive and offensive strategies. In this paper, we discuss the unique and common features of phage resistance mechanisms and their role in global biodiversity. Finally, the commonalities between defense mechanisms suggest avenues for the discovery of novel forms of these mechanisms based on their evolutionary traits.

  11. METHOD AND LOCATION OF GROUND WATER SAMPLING: IMPACT ON ATTENUATION FACTORS FOR ASSESSING IMPACT ON VAPOR INTRUSION

    EPA Science Inventory

    The Draft EPA Subsurface Vapor Intrusion Guidance Document was established to "address the incremental increases in exposures and risks from subsurface contaminants that my be intruding into indoor air". The document utilizes attenuation factors based on indoor air/soil gas or i...

  12. Targeting mammalian organelles with internalizing phage (iPhage) libraries

    PubMed Central

    Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

  13. Kinetics of filamentous phage assembly

    NASA Astrophysics Data System (ADS)

    Ploss, Martin; Kuhn, Andreas

    2010-12-01

    Filamentous phages release their progeny particles by a secretory process without lysing the bacterial cell. By this process about 6 viral particles per min are secreted from each cell. We show here that when the major coat protein (gp8) is provided from a plasmid we observe a phage progeny production rate depending on the induction of gp8 by IPTG. We also show that a transfection of Escherichia coli lacking F-pili is observed using a mutant of M13 that carries an ampicillin resistance gene, and phage particles are secreted in the absence of an F-plasmid. Extruding phage was visualized by atomic force microscopy (AFM) and by transmission electron microscopy (TEM) using gold-labeled antibodies to the major coat protein.

  14. Self-attenuation correction factors for bioindicators measured by γ spectrometry for energies <100 keV

    NASA Astrophysics Data System (ADS)

    Manduci, L.; Tenailleau, L.; Trolet, J. L.; De Vismes, A.; Lopez, G.; Piccione, M.

    2010-01-01

    The mass attenuation coefficients for a number of marine and terrestrial bioindicators were measured using γ spectrometry for energies between 22 and 80 keV. These values were then used to find the correction factor k for the apparent radioactivity. The experimental results were compared with a Monte Carlo simulation performed using PENELOPE in order to evaluate the reliability of the simplified calculation and to determine the correction factors.

  15. Inhibition of Epidermal Growth Factor Receptor Improves Myelination and Attenuates Tissue Damage of Spinal Cord Injury.

    PubMed

    Zhang, Si; Ju, Peijun; Tjandra, Editha; Yeap, Yeeshan; Owlanj, Hamed; Feng, Zhiwei

    2016-10-01

    Preventing demyelination and promoting remyelination of denuded axons are promising therapeutic strategies for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibition was reported to benefit the neural functional recovery and the axon regeneration after SCI. However, its role in de- and remyelination of axons in injured spinal cord is unclear. In the present study, we evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. We found that expression of myelin basic protein (MBP) in the injured spinal cords of PD treated mice was remarkably elevated. The density of glial precursor cells and oligodendrocytes (OLs) was increased and the cell apoptosis in lesions was attenuated after PD168393 treatment. Moreover, PD168393 treatment reduced both the numbers of OX42 + microglial cells and glial fibrillary acidic protein + astrocytes in damaged area of spinal cords. We thus conclude that the therapeutic effects of EGFR inhibition after SCI involves facilitating remyelination of the injured spinal cord, increasing of oligodendrocyte precursor cells and OLs, as well as suppressing the activation of astrocytes and microglia/macrophages. PMID:26883518

  16. Bacteria-phage interactions in natural environments.

    PubMed

    Díaz-Muñoz, Samuel L; Koskella, Britt

    2014-01-01

    Phages are considered the most abundant and diverse biological entities on Earth and are notable not only for their sheer abundance, but also for their influence on bacterial hosts. In nature, bacteria-phage relationships are complex and have far-reaching consequences beyond particular pairwise interactions, influencing everything from bacterial virulence to eukaryotic fitness to the carbon cycle. In this review, we examine bacteria and phage distributions in nature first by highlighting biogeographic patterns and nonhost environmental influences on phage distribution, then by considering the ways in which phages and bacteria interact, emphasizing phage life cycles, bacterial responses to phage infection, and the complex patterns of phage host specificity. Finally, we discuss phage impacts on bacterial abundance, genetics, and physiology, and further aim to clarify distinctions between current theoretical models and point out areas in need of future research. PMID:25131402

  17. Advance in phage display technology for bioanalysis.

    PubMed

    Tan, Yuyu; Tian, Tian; Liu, Wenli; Zhu, Zhi; J Yang, Chaoyong

    2016-06-01

    Phage display technology has emerged as a powerful tool for target gene expression and target-specific ligand selection. It is widely used to screen peptides, proteins and antibodies with the advantages of simplicity, high efficiency and low cost. A variety of targets, including ions, small molecules, inorganic materials, natural and biological polymers, nanostructures, cells, bacteria, and even tissues, have been demonstrated to generate specific binding ligands by phage display. Phages and target-specific ligands screened by phage display have been widely used as affinity reagents in therapeutics, diagnostics and biosensors. In this review, comparisons of different types of phage display systems are first presented. Particularly, microfluidic-based phage display, which enables screening with high throughput, high efficiency and integration, is highlighted. More importantly, we emphasize the advances in biosensors based on phages or phage-derived probes, including nonlytic phages, lytic phages, peptides or proteins screened by phage display, phage assemblies and phage-nanomaterial complexes. However, more efficient and higher throughput phage display methods are still needed to meet an explosion in demand for bioanalysis. Furthermore, screening of cyclic peptides and functional peptides will be the hotspot in bioanalysis. PMID:27061133

  18. SPR Biosensor for the Detection of L. monocytogenes using Phage Displayed Antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Whole cells of Listeria monocytogenes were detected with a compact, surface plasmon resonance (SPR) sensor using a phage-displayed scFv antibody to the virulence factor ActA for biorecognition. Phage Lm P4:A8, expressing the scFv antibody fused to the pIII surface protein was immobilized to the se...

  19. Comparative genomics and functional analysis of the 936 group of lactococcal Siphoviridae phages

    PubMed Central

    Murphy, James; Bottacini, Francesca; Mahony, Jennifer; Kelleher, Philip; Neve, Horst; Zomer, Aldert; Nauta, Arjen; van Sinderen, Douwe

    2016-01-01

    Genome sequencing and comparative analysis of bacteriophage collections has greatly enhanced our understanding regarding their prevalence, phage-host interactions as well as the overall biodiversity of their genomes. This knowledge is very relevant to phages infecting Lactococcus lactis, since they constitute a significant risk factor for dairy fermentations. Of the eighty four lactococcal phage genomes currently available, fifty five belong to the so-called 936 group, the most prevalent of the ten currently recognized lactococcal phage groups. Here, we report the genetic characteristics of a new collection of 936 group phages. By combining these genomes to those sequenced previously we determined the core and variable elements of the 936 genome. Genomic variation occurs across the 936 phage genome, such as genetic elements that (i) lead to a +1 translational frameshift resulting in the formation of additional structures on the phage tail, (ii) specify a double neck passage structure, and (iii) encode packaging module-associated methylases. Hierarchical clustering of the gene complement of the 936 group phages and nucleotide alignments allowed grouping of the ninety 936 group phages into distinct clusters, which in general appear to correspond with their geographical origin. PMID:26892066

  20. Development of an engineered bioluminescent reporter phage for detection of bacterial blight of crucifers.

    PubMed

    Schofield, David A; Bull, Carolee T; Rubio, Isael; Wechter, W Patrick; Westwater, Caroline; Molineux, Ian J

    2012-05-01

    Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant "light"-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis. PMID:22427491

  1. Development of an Engineered Bioluminescent Reporter Phage for Detection of Bacterial Blight of Crucifers

    PubMed Central

    Bull, Carolee T.; Rubio, Isael; Wechter, W. Patrick; Westwater, Caroline; Molineux, Ian J.

    2012-01-01

    Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant “light”-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis. PMID:22427491

  2. Attenuated brain-derived neurotrophic factor and hypertrophic remodelling: the SABPA study.

    PubMed

    Smith, A J; Malan, L; Uys, A S; Malan, N T; Harvey, B H; Ziemssen, T

    2015-01-01

    Brain-derived neurotrophic factor (BDNF) has been linked to neurological pathologies, but its role in cardiometabolic disturbances is limited. We aimed to assess the association between serum BDNF levels and structural endothelial dysfunction (ED) as determined by cross-sectional wall area (CSWA) and albumin/creatinine ratio (ACR) in black Africans. Ambulatory blood pressure (BP) and ultrasound CSWA values were obtained from 82 males and 90 females. Fasting blood and 8 h overnight urine samples were collected to determine serum BDNF and cardiometabolic risk markers, that is, glycated haemoglobin (HbA1c), lipids, inflammation and ACR. BDNF median split × gender interaction effects for structural ED justified stratification of BDNF into low and high (⩽/>1.37 ng ml(-1)) gender groups. BDNF values (0.86-1.98 ng ml(-1)) were substantially lower than reference ranges (6.97-42.6 ng ml(-1)) in the African gender cohort, independent of age and body mass index. No relationship was revealed between BDNF and renal function and was opposed by an inverse relationship between BDNF and CSWA (r=-0.17; P=0.03) in the African cohort. Linear regression analyses revealed a positive relationship between systolic BP and structural remodelling in the total cohort and low-BDNF gender groups. In the high-BDNF females, HbA1C was associated with structural remodelling. Attenuated or possible downregulated BDNF levels were associated with hypertrophic remodelling, and may be a compensatory mechanism for the higher BP in Africans. In addition, metabolic risk and hypertrophic remodelling in women with high BDNF underpin different underlying mechanisms for impaired neurotrophin homeostasis in men and women. PMID:24898921

  3. Early Systemic Granulocyte-Colony Stimulating Factor Treatment Attenuates Neuropathic Pain after Peripheral Nerve Injury

    PubMed Central

    Lee, Yun-Lin; Chen, Jin-Chung; Wang, Hung-Li; Yang, Yi-Ling; Cheng, Mei-Yun; Liao, Ming-Feng; Ro, Long-Sun

    2012-01-01

    Recent studies have shown that opioid treatment can reduce pro-inflammatory cytokine production and counteract various neuropathic pain syndromes. Granulocyte colony-stimulating factor (G-CSF) can promote immune cell differentiation by increasing leukocytes (mainly opioid-containing polymorphonuclear (PMN) cells), suggesting a potential beneficial role in treating chronic pain. This study shows the effectiveness of exogenous G-CSF treatment (200 µg/kg) for alleviating thermal hyperalgesia and mechanical allodynia in rats with chronic constriction injury (CCI), during post-operative days 1–25, compared to that of vehicle treatment. G-CSF also increases the recruitment of opioid-containing PMN cells into the injured nerve. After CCI, single administration of G-CSF on days 0, 1, and 2, but not on day 3, relieved thermal hyperalgesia, which indicated that its effect on neuropathic pain had a therapeutic window of 0–48 h after nerve injury. CCI led to an increase in the levels of interleukin-6 (IL-6) mRNA and tumor necrosis factor-α (TNF-α) protein in the dorsal root ganglia (DRG). These high levels of IL-6 mRNA and TNF-α were suppressed by a single administration of G-CSF 48–144 h and 72–144 h after CCI, respectively. Furthermore, G-CSF administered 72–144 h after CCI suppressed the CCI-induced upregulation of microglial activation in the ipsilateral spinal dorsal horn, which is essential for sensing neuropathic pain. Moreover, the opioid receptor antagonist naloxone methiodide (NLXM) reversed G-CSF-induced antinociception 3 days after CCI, suggesting that G-CSF alleviates hyperalgesia via opioid/opioid receptor interactions. These results suggest that an early single systemic injection of G-CSF alleviates neuropathic pain via activation of PMN cell-derived endogenous opioid secretion to activate opioid receptors in the injured nerve, downregulate IL-6 and TNF-α inflammatory cytokines, and attenuate microglial activation in the spinal dorsal horn. This

  4. Membrane fusion during phage lysis.

    PubMed

    Rajaure, Manoj; Berry, Joel; Kongari, Rohit; Cahill, Jesse; Young, Ry

    2015-04-28

    In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of μm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG. PMID:25870259

  5. Membrane fusion during phage lysis

    PubMed Central

    Berry, Joel; Kongari, Rohit; Cahill, Jesse; Young, Ry

    2015-01-01

    In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of μm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG. PMID:25870259

  6. Attenuation of arsenic in a karst subterranean stream and correlation with geochemical factors: a case study at Lihu, South China.

    PubMed

    Zhang, Liankai; Yang, Hui; Tang, Jiansheng; Qin, Xiaoqun; Yu, Au Yik

    2014-11-01

    Arsenic (As) pollutants generated by human activities in karst areas flow into subterranean streams and contaminate groundwater easily because of the unique hydrogeological characteristics of karst areas. To elucidate the reaction mechanisms of arsenic in karst subterranean streams, physical-chemical analysis was conducted by an inductively coupled plasma mass spectrometer and an X-ray fluorescence spectrometer. The results show that inorganic species account for most of the total arsenic, whereas organic arsenic is not detected or occurs in infinitesimal amounts. As(III) accounts for 51.0%±9.9% of the total inorganic arsenic. Arsenic attenuation occurs and the attenuation rates of total As, As(III) and As(V) in the Lihu subterranean stream are 51%, 36% and 59%, respectively. To fully explain the main geochemical factors influencing arsenic attenuation, SPSS 13.0 and CANOCO 4.5 bundled with CanoDraw for Windows were used for simple statistical analysis and redundancy analysis (RDA). Eight main factors, i.e., sediment iron (SFe), sediment aluminum (SAl), sediment calcium (SCa), sediment organic matter (SOM), sediment manganese (SMn), water calcium (WCa(2+)), water magnesium (WMg(2+)), and water bicarbonate ion (WHCO3(-)) were extracted from thirteen indicators. Their impacts on arsenic content rank as: SFe>SCa>WCa(2+)>SAl>WHCO3(-)>SMn>SOM>WMg(2+). Of these factors, SFe, SAl, SCa, SOM, SMn, WMg(2+) and WCa(2+) promote arsenic attenuation, whereas WHCO3(-) inhibits it. Further investigation revealed that the redox potential (Eh) and pH are adverse to arsenic removal. The dramatic distinction between karst and non-karst terrain is that calcium and bicarbonate are the primary factors influencing arsenic migration in karst areas due to the high calcium concentration and alkalinity of karst water. PMID:25458676

  7. Transposable Phage Mu.

    PubMed

    Harshey, Rasika M

    2014-10-01

    Transposable phage Mu has played a major role in elucidating the mechanism of movement of mobile DNA elements. The high efficiency of Mu transposition has facilitated a detailed biochemical dissection of the reaction mechanism, as well as of protein and DNA elements that regulate transpososome assembly and function. The deduced phosphotransfer mechanism involves in-line orientation of metal ion-activated hydroxyl groups for nucleophilic attack on reactive diester bonds, a mechanism that appears to be used by all transposable elements examined to date. A crystal structure of the Mu transpososome is available. Mu differs from all other transposable elements in encoding unique adaptations that promote its viral lifestyle. These adaptations include multiple DNA (enhancer, SGS) and protein (MuB, HU, IHF) elements that enable efficient Mu end synapsis, efficient target capture, low target specificity, immunity to transposition near or into itself, and efficient mechanisms for recruiting host repair and replication machineries to resolve transposition intermediates. MuB has multiple functions, including target capture and immunity. The SGS element promotes gyrase-mediated Mu end synapsis, and the enhancer, aided by HU and IHF, participates in directing a unique topological architecture of the Mu synapse. The function of these DNA and protein elements is important during both lysogenic and lytic phases. Enhancer properties have been exploited in the design of mini-Mu vectors for genetic engineering. Mu ends assembled into active transpososomes have been delivered directly into bacterial, yeast, and human genomes, where they integrate efficiently, and may prove useful for gene therapy. PMID:26104374

  8. Factors affecting survival of bacteriophage on tomato leaf surfaces.

    PubMed

    Iriarte, F B; Balogh, B; Momol, M T; Smith, L M; Wilson, M; Jones, J B

    2007-03-01

    The ability of bacteriophage to persist in the phyllosphere for extended periods is limited by many factors, including sunlight irradiation, especially in the UV zone, temperature, desiccation, and exposure to copper bactericides. The effects of these factors on persistence of phage and formulated phage (phage mixed with skim milk) were evaluated. In field studies, copper caused significant phage reduction if applied on the day of phage application but not if applied 4 or 7 days in advance. Sunlight UV was evaluated for detrimental effects on phage survival on tomato foliage in the field. Phage was applied in the early morning, midmorning, early afternoon, and late evening, while UVA plus UVB irradiation and phage populations were monitored. The intensity of UV irradiation positively correlated with phage population decline. The protective formulation reduced the UV effect. In order to demonstrate direct effects of UV, phage suspensions were exposed to UV irradiation and assayed for effectiveness against bacterial spot of tomato. UV significantly reduced phage ability to control bacterial spot. Ambient temperature had a pronounced effect on nonformulated phage but not on formulated phages. The effects of desiccation and fluorescent light illumination on phage were investigated. Desiccation caused a significant but only slight reduction in phage populations after 60 days, whereas fluorescent light eliminated phages within 2 weeks. The protective formulation eliminated the reduction caused by both of these factors. Phage persistence was dramatically affected by UV, while the other factors had less pronounced effects. Formulated phage reduced deleterious effects of the studied environmental factors. PMID:17259361

  9. Phage therapy—constraints and possibilities

    PubMed Central

    2014-01-01

    The rise of antibiotic-resistant bacterial strains, causing intractable infections, has resulted in an increased interest in phage therapy. Phage therapy preceded antibiotic treatment against bacterial infections and involves the use of bacteriophages, bacterial viruses, to fight bacteria. Virulent phages are abundant and have proven to be very effective in vitro, where they in most cases lyse any bacteria within the hour. Clinical trials on animals and humans show promising results but also that the treatments are not completely effective. This is partly due to the studies being carried out with few phages, and with limited experimental groups, but also the fact that phage therapy has limitations in vivo. Phages are large compared with small antibiotic molecules, and each phage can only infect one or a few bacterial strains. A very large number of different phages are needed to treat infections as these are caused by genetically different strains of bacteria. Phages are effective only if enough of them can reach the bacteria and increase in number in situ. Taken together, this entails high demands on resources for the construction of phage libraries and the testing of individual phages. The effectiveness and host range must be characterized, and immunological risks must be assessed for every single phage. PMID:24678769

  10. Dynamic phages in the swine gut ecosystem

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phages are important drivers of ecosystem functions, yet they are often overlooked in gut microbiome studies. Inclusion of phages in gut microbiome analyses is essential to deciphering complex gut ecology under both normal and disturbed conditions. To assess the effect of antibiotics on phage activi...

  11. Nitrogen attenuation of terrestrial carbon cycle response to global environmental factors

    USGS Publications Warehouse

    Jain, A.A.; Yang, Xiaojuan; Kheshgi, H.; McGuire, Anthony; Post, W.; Kicklighter, David W.

    2009-01-01

    Nitrogen cycle dynamics have the capacity to attenuate the magnitude of global terrestrial carbon sinks and sources driven by CO2 fertilization and changes in climate. In this study, two versions of the terrestrial carbon and nitrogen cycle components of the Integrated Science Assessment Model (ISAM) are used to evaluate how variation in nitrogen availability influences terrestrial carbon sinks and sources in response to changes over the 20th century in global environmental factors including atmospheric CO2 concentration, nitrogen inputs, temperature, precipitation and land use. The two versions of ISAM vary in their treatment of nitrogen availability: ISAM-NC has a terrestrial carbon cycle model coupled to a fully dynamic nitrogen cycle while ISAM-C has an identical carbon cycle model but nitrogen availability is always in sufficient supply. Overall, the two versions of the model estimate approximately the same amount of global mean carbon uptake over the 20th century. However, comparisons of results of ISAM-NC relative to ISAM-C reveal that nitrogen dynamics: (1) reduced the 1990s carbon sink associated with increasing atmospheric CO2 by 0.53 PgC yr−1 (1 Pg = 1015g), (2) reduced the 1990s carbon source associated with changes in temperature and precipitation of 0.34 PgC yr−1 in the 1990s, (3) an enhanced sink associated with nitrogen inputs by 0.26 PgC yr−1, and (4) enhanced the 1990s carbon source associated with changes in land use by 0.08 PgC yr−1 in the 1990s. These effects of nitrogen limitation influenced the spatial distribution of the estimated exchange of CO2 with greater sink activity in high latitudes associated with climate effects and a smaller sink of CO2 in the southeastern United States caused by N limitation associated with both CO2 fertilization and forest regrowth. These results indicate that the dynamics of nitrogen availability are important to consider in assessing the spatial distribution and temporal dynamics of terrestrial carbon

  12. Nitrogen attenuation of terrestrial carbon cycle response to global environmental factors

    SciTech Connect

    Jain, Atul; Yang, Xiaojuan; Kheshgi, Haroon; Mcguire, David; Post, Wilfred M

    2009-01-01

    Nitrogen cycle dynamics have the capacity to attenuate the magnitude of global terrestrial carbon sinks and sources driven by CO2 fertilization and changes in climate. In this study, two versions of the terrestrial carbon and nitrogen cycle components of the Integrated Science Assessment Model (ISAM) are used to evaluate how variation in nitrogen availability influences terrestrial carbon sinks and sources in response to changes over the 20th century in global environmental factors including atmospheric CO2 concentration, nitrogen inputs, temperature, precipitation and land use. The two versions of ISAM vary in their treatment of nitrogen availability: ISAM-NC has a terrestrial carbon cycle model coupled to a fully dynamic nitrogen cycle while ISAM-C has an identical carbon cycle model but nitrogen availability is always in sufficient supply. Overall, the two versions of the model estimate approximately the same amount of global mean carbon uptake over the 20th century. However, comparisons of results of ISAM-NC relative to ISAM-C reveal that nitrogen dynamics: (1) reduced the 1990s carbon sink associated with increasing atmospheric CO2 by 0.53 PgC yr1 (1 Pg = 1015g), (2) reduced the 1990s carbon source associated with changes in temperature and precipitation of 0.34 PgC yr1 in the 1990s, (3) an enhanced sink associated with nitrogen inputs by 0.26 PgC yr1, and (4) enhanced the 1990s carbon source associated with changes in land use by 0.08 PgC yr1 in the 1990s. These effects of nitrogen limitation influenced the spatial distribution of the estimated exchange of CO2 with greater sink activity in high latitudes associated with climate effects and a smaller sink of CO2 in the southeastern United States caused by N limitation associated with both CO2 fertilization and forest regrowth. These results indicate that the dynamics of nitrogen availability are important to consider in assessing the spatial distribution and temporal dynamics of terrestrial carbon sources and

  13. Nitrogen attenuation of terrestrial carbon cycle response to global environmental factors

    NASA Astrophysics Data System (ADS)

    Jain, Atul; Yang, Xiaojuan; Kheshgi, Haroon; McGuire, A. David; Post, Wilfred; Kicklighter, David

    2009-12-01

    Nitrogen cycle dynamics have the capacity to attenuate the magnitude of global terrestrial carbon sinks and sources driven by CO2 fertilization and changes in climate. In this study, two versions of the terrestrial carbon and nitrogen cycle components of the Integrated Science Assessment Model (ISAM) are used to evaluate how variation in nitrogen availability influences terrestrial carbon sinks and sources in response to changes over the 20th century in global environmental factors including atmospheric CO2 concentration, nitrogen inputs, temperature, precipitation and land use. The two versions of ISAM vary in their treatment of nitrogen availability: ISAM-NC has a terrestrial carbon cycle model coupled to a fully dynamic nitrogen cycle while ISAM-C has an identical carbon cycle model but nitrogen availability is always in sufficient supply. Overall, the two versions of the model estimate approximately the same amount of global mean carbon uptake over the 20th century. However, comparisons of results of ISAM-NC relative to ISAM-C reveal that nitrogen dynamics: (1) reduced the 1990s carbon sink associated with increasing atmospheric CO2 by 0.53 PgC yr-1 (1 Pg = 1015g), (2) reduced the 1990s carbon source associated with changes in temperature and precipitation of 0.34 PgC yr-1 in the 1990s, (3) an enhanced sink associated with nitrogen inputs by 0.26 PgC yr-1, and (4) enhanced the 1990s carbon source associated with changes in land use by 0.08 PgC yr-1 in the 1990s. These effects of nitrogen limitation influenced the spatial distribution of the estimated exchange of CO2 with greater sink activity in high latitudes associated with climate effects and a smaller sink of CO2 in the southeastern United States caused by N limitation associated with both CO2 fertilization and forest regrowth. These results indicate that the dynamics of nitrogen availability are important to consider in assessing the spatial distribution and temporal dynamics of terrestrial carbon sources

  14. Synthetic Phage for Tissue Regeneration

    PubMed Central

    Merzlyak, Anna; Lee, Seung-Wuk

    2014-01-01

    Controlling structural organization and signaling motif display is of great importance to design the functional tissue regenerating materials. Synthetic phage, genetically engineered M13 bacteriophage has been recently introduced as novel tissue regeneration materials to display a high density of cell-signaling peptides on their major coat proteins for tissue regeneration purposes. Structural advantages of their long-rod shape and monodispersity can be taken together to construct nanofibrous scaffolds which support cell proliferation and differentiation as well as direct orientation of their growth in two or three dimensions. This review demonstrated how functional synthetic phage is designed and subsequently utilized for tissue regeneration that offers potential cell therapy. PMID:24991085

  15. Alternative Pathway Inhibition by Exogenous Factor H Fails to Attenuate Inflammation and Vascular Leakage in Experimental Pneumococcal Sepsis in Mice.

    PubMed

    van der Maten, Erika; van Selm, Saskia; Langereis, Jeroen D; Bootsma, Hester J; van Opzeeland, Fred J H; de Groot, Ronald; de Jonge, Marien I; van der Flier, Michiel

    2016-01-01

    Streptococcus pneumoniae is a common cause of sepsis. Effective complement activation is an important component of host defence against invading pathogens, whilst excessive complement activation has been associated with endothelial dysfunction and organ damage. The alternative pathway amplification loop is important for the enhancement of complement activation. Factor H is a key negative regulator of the alternative pathway amplification loop and contributes to tight control of complement activation. We assessed the effect of inhibition of the alternative pathway on sepsis associated inflammation and disease severity using human factor H treatment in a clinically relevant mice model of pneumococcal sepsis. Mice were infected intravenously with live Streptococcus pneumoniae. At the first clinical signs of infection, 17 hours post-infection, mice were treated with ceftriaxone antibiotic. At the same time purified human factor H or in controls PBS was administered. Treatment with human factor H did not attenuate disease scores, serum pro-inflammatory cytokines, or vascular permeability and did not significantly affect C3 and C3a production at 26 h post-infection. Therefore, we conclude that inhibition of the alternative complement pathway by exogenous human factor H fails to attenuate inflammation and vascular leakage at a clinically relevant intervention time point in pneumococcal sepsis in mice. PMID:26872035

  16. Alternative Pathway Inhibition by Exogenous Factor H Fails to Attenuate Inflammation and Vascular Leakage in Experimental Pneumococcal Sepsis in Mice

    PubMed Central

    van der Maten, Erika; van Selm, Saskia; Langereis, Jeroen D.; Bootsma, Hester J.; van Opzeeland, Fred J. H.; de Groot, Ronald; de Jonge, Marien I.; van der Flier, Michiel

    2016-01-01

    Streptococcus pneumoniae is a common cause of sepsis. Effective complement activation is an important component of host defence against invading pathogens, whilst excessive complement activation has been associated with endothelial dysfunction and organ damage. The alternative pathway amplification loop is important for the enhancement of complement activation. Factor H is a key negative regulator of the alternative pathway amplification loop and contributes to tight control of complement activation. We assessed the effect of inhibition of the alternative pathway on sepsis associated inflammation and disease severity using human factor H treatment in a clinically relevant mice model of pneumococcal sepsis. Mice were infected intravenously with live Streptococcus pneumoniae. At the first clinical signs of infection, 17 hours post-infection, mice were treated with ceftriaxone antibiotic. At the same time purified human factor H or in controls PBS was administered. Treatment with human factor H did not attenuate disease scores, serum pro-inflammatory cytokines, or vascular permeability and did not significantly affect C3 and C3a production at 26 h post-infection. Therefore, we conclude that inhibition of the alternative complement pathway by exogenous human factor H fails to attenuate inflammation and vascular leakage at a clinically relevant intervention time point in pneumococcal sepsis in mice. PMID:26872035

  17. Monsoon variability of ultraviolet radiation (UVR) attenuation and bio-optical factors in the Asian tropical coral-reef waters

    NASA Astrophysics Data System (ADS)

    Mizubayashi, Keiko; Kuwahara, Victor S.; Segaran, Thirukanthan C.; Zaleha, Kassim; Effendy, A. W. M.; Kushairi, M. R. M.; Toda, Tatsuki

    2013-07-01

    The East coast of Peninsular Malaysia is strongly influenced by the North-East (NE) monsoon, and may significantly influence the optical environment of coral-reef ecosystems. However, our knowledge of temporal variability, including episodic events, of environmental factors in Asian tropical regions is still limited. The objectives of this study were to (1) observe temporal variability in ultraviolet radiation (UVR) and photosynthetically active radiation (PAR) attenuation and (2) determine the bio-optical factors regulating the optical environment in shallow coral-reef waters. Downwelling UVR and PAR irradiance and in situ bio-optical factors were measured monthly near Bidong Island on the East coast of Peninsular Malaysia from June 2010 to June 2011. The NE monsoon was recognized between November 2010 and January 2011. The highest diffuse attenuation coefficient at 305 nm was 2.05 ± 0.03 m-1 in a coral-reef area on December 2010. The most significant bio-optical factor at 305, 380, 440 nm during the NE monsoon season was CDOM (89 ± 8% at 305 nm, 84 ± 9% at 380 nm and 49 ± 17% at 440 nm). All UVR attenuation coefficients showed significant correlations with the CDOM absorption coefficients (aCDOM). CDOM with relatively low S275-295 during the NE monsoon season (0.0177 ± 0.0020 nm-1) suggests terrestrial sources, which is also supported by the correlation between salinity and aCDOM(305). A significant correlation between S275-295 and the carbon specific absorbance coefficient (a*(305)) suggest the potential to measure DOC optically in these waters. The high CDOM during the NE monsoon season may have an important role to reduce harmful UVR exposure reaching benthic communities.

  18. Apocynin Attenuates Cardiac Injury in Type 4 Cardiorenal Syndrome via Suppressing Cardiac Fibroblast Growth Factor-2 With Oxidative Stress Inhibition

    PubMed Central

    Liu, Yang; Liu, Yu; Liu, Xun; Chen, Jie; Zhang, Kun; Huang, Feifei; Wang, Jing-Feng; Tang, Wanchun; Huang, Hui

    2015-01-01

    Background Type 4 cardiorenal syndrome (CRS) refers to the cardiac injury induced by chronic kidney disease. We aimed to assess oxidative stress and cardiac injury in patients with type 4 CRS, determine whether the antioxidant apocynin attenuated cardiac injury in rats with type 4 CRS, and explore potential mechanisms. Methods and Results A cross-sectional study was conducted among patients with type 4 CRS (n=17) and controls (n=16). Compared with controls, patients with type 4 CRS showed elevated oxidative stress, which was significantly correlated with cardiac hypertrophy and decreased ejection fraction. In vivo study, male Sprague-Dawley rats underwent 5/6 subtotal nephrectomy and sham surgery, followed with apocynin or vehicle treatment for 8 weeks. Eight weeks after surgery, the 5/6 subtotal nephrectomy rats mimicked type 4 CRS, showing increased serum creatinine, cardiac hypertrophy and fibrosis, and decreased ejection fraction compared with sham-operated animals. Cardiac malondialdehyde, NADPH oxidase activity, fibroblast growth factor-2, and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation increased significantly in the 5/6 subtotal nephrectomy rats. These changes were significantly attenuated by apocynin. In vitro study showed that apocynin reduced angiotensin II–induced NADPH oxidase–dependent oxidative stress, upregulation of fibroblast growth factor-2 and fibrosis biomarkers, and ERK1/2 phosphorylation in cardiac fibroblasts. Importantly, the ERK1/2 inhibitor U0126 reduced the upregulation of fibroblast growth factor-2 and fibrosis biomarkers in angiotensin II–treated fibroblasts. Conclusions Oxidative stress is a candidate mediator for type 4 CRS. Apocynin attenuated cardiac injury in type 4 CRS rats via inhibiting NADPH oxidase–dependent oxidative stress-activated ERK1/2 pathway and subsequent fibroblast growth factor-2 upregulation. Our study added evidence to the beneficial effect of apocynin in type 4 CRS. PMID:26109504

  19. Protein and Antibody Engineering by Phage Display.

    PubMed

    Frei, J C; Lai, J R

    2016-01-01

    Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

  20. Thioredoxin is required for filamentous phage assembly.

    PubMed Central

    Russel, M; Model, P

    1985-01-01

    Sequence comparisons show that the fip gene product of Escherichia coli, which is required for filamentous phage assembly, is thioredoxin. Thioredoxin serves as a cofactor for reductive processes in many cell types and is a constituent of phage T7 DNA polymerase. The fip-1 mutation makes filamentous phage and T7 growth temperature sensitive in cells that carry it. The lesion lies within a highly conserved thioredoxin active site. Thioredoxin reductase (NADPH), as well as thioredoxin, is required for efficient filamentous phage production. Mutant phages defective in phage gene I are particularly sensitive to perturbations in the fip-thioredoxin system. A speculative model is presented in which thioredoxin reductase, thioredoxin, and the gene I protein interact to drive an engine for filamentous phage assembly. Images PMID:3881756

  1. Phage Therapy: Eco-Physiological Pharmacology

    PubMed Central

    Abedon, Stephen T.

    2014-01-01

    Bacterial virus use as antibacterial agents, in the guise of what is commonly known as phage therapy, is an inherently physiological, ecological, and also pharmacological process. Physiologically we can consider metabolic properties of phage infections of bacteria and variation in those properties as a function of preexisting bacterial states. In addition, there are patient responses to pathogenesis, patient responses to phage infections of pathogens, and also patient responses to phage virions alone. Ecologically, we can consider phage propagation, densities, distribution (within bodies), impact on body-associated microbiota (as ecological communities), and modification of the functioning of body “ecosystems” more generally. These ecological and physiological components in many ways represent different perspectives on otherwise equivalent phenomena. Comparable to drugs, one also can view phages during phage therapy in pharmacological terms. The relatively unique status of phages within the context of phage therapy as essentially replicating antimicrobials can therefore result in a confluence of perspectives, many of which can be useful towards gaining a better mechanistic appreciation of phage therapy, as I consider here. Pharmacology more generally may be viewed as a discipline that lies at an interface between organism-associated phenomena, as considered by physiology, and environmental interactions as considered by ecology. PMID:25031881

  2. Recombinant phage probes for Listeria monocytogenes

    NASA Astrophysics Data System (ADS)

    Carnazza, S.; Gioffrè, G.; Felici, F.; Guglielmino, S.

    2007-10-01

    Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

  3. Insertion of host DNA into PVL-encoding phages of the Staphylococcus aureus lineage ST80 by intra-chromosomal recombination.

    PubMed

    Wirtz, Christiane; Witte, Wolfgang; Wolz, Christiane; Goerke, Christiane

    2010-10-25

    Temperate bacteriophages play a critical role in the pathogenicity of the human pathogen Staphylococcus aureus by mediating positive lysogenic conversion for different virulence factors such as Panton-Valentine leukocidin (PVL) or by interrupting chromosomal virulence genes. PVL-encoding phages are integrated in the S. aureus genome within a conserved ORF which is surrounded by a cluster of tandemly repeated genes. Here we demonstrate that in S. aureus clonal complex ST80 strains PVL-phage induction led to the acquisition of host DNA into the phage genome probably due to a homologous recombination event between direct repeats of the two paralogous genes adjacent to the phage integration site. Phage excision was accompanied by an additional chromosomal deletion in this region. This so far unrecognized mechanism of DNA uptake into the phage genome may play an important role in the co-evolution of phages and bacteria. PMID:20708208

  4. Experimental investigation of the factors influencing temperature dependence of radiation-induced attenuation in optical fiber

    NASA Astrophysics Data System (ADS)

    Jin, Jing; Xu, Raomei; Liu, Jixun; Song, Ningfang

    2014-03-01

    The effects of transmission wavelength, total dose and light source power on temperature dependence of radiation-induced attenuation (RIA) in Ge-P co-doped fibers were investigated. Three fibers irradiated up to total dose of 100 Gy and 10,000 Gy were used as test samples. A test system for temperature dependence of RIA was built up. The influence of transmission wavelength, total dose and light power on temperature sensitivity and linearity of RIA in three irradiated fibers were researched. The test results show that temperature sensitivity and linearity of RIA in optical fibers could be improved by adjusting total dose and selecting transmission wavelength. The light source power does not have obvious influence on temperature sensitivity and linearity. The Ge-P co-doped fiber at 850 nm transmission wavelength with higher total dose is a very promising candidate for fiber-optic temperature sensor.

  5. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1.

    PubMed

    Dyson, Zoe A; Seviour, Robert J; Tucci, Joseph; Petrovski, Steve

    2016-01-01

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA. PMID:27313312

  6. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1

    PubMed Central

    Dyson, Zoe A.; Seviour, Robert J.; Tucci, Joseph

    2016-01-01

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa. The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA. PMID:27313312

  7. Bacteriophages with potential to inactivate Salmonella Typhimurium: Use of single phage suspensions and phage cocktails.

    PubMed

    Pereira, Carla; Moreirinha, Catarina; Lewicka, Magdalena; Almeida, Paulo; Clemente, Carla; Cunha, Ângela; Delgadillo, Ivonne; Romalde, Jésus L; Nunes, Maria L; Almeida, Adelaide

    2016-07-15

    The aim of this study was to compare the dynamics of three previously isolated bacteriophages (or phages) individually (phSE-1, phSE-2 and phSE-5) or combined in cocktails of two or three phages (phSE-1/phSE-2, phSE-1/phSE-5, phSE-2/phSE-5 and phSE-1/phSE-2/phSE-5) to control Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) in order to evaluate their potential application during depuration. Phages were assigned to the family Siphoviridae and revealed identical restriction digest profiles, although they showed a different phage adsorption, host range, burst size, explosion time and survival in seawater. The three phages were effective against S. Typhimurium (reduction of ∼2.0 log CFU/mL after 4h treatment). The use of cocktails was not significantly more effective than the use of single phages. A big fraction of the remained bacteria are phage-resistant mutants (frequency of phage-resistant mutants 9.19×10(-5)-5.11×10(-4)) but phage- resistant bacterial mutants was lower for the cocktail phages than for the single phage suspensions and the phage phSE-1 presented the highest rate of resistance and phage phSE-5 the lowest one. The spectral changes of S. Typhimurium resistant and phage-sensitive cells were compared and revealed relevant differences for peaks associated to amide I (1620cm(-1)) and amide II (1515cm(-1)) from proteins and from carbohydrates and phosphates region (1080-1000cm(-1)). Despite the similar efficiency of individual phages, the development of lower resistance indicates that phage cocktails might be the most promising choice to be used during the bivalve depuration to control the transmission of salmonellosis. PMID:27126773

  8. Complete genome sequence analysis of two Pseudomonas plecoglossicida phages, potential therapeutic agents.

    PubMed

    Kawato, Yasuhiko; Yasuike, Motoshige; Nakamura, Yoji; Shigenobu, Yuya; Fujiwara, Atushi; Sano, Motohiko; Nakai, Toshihiro

    2015-02-01

    Pseudomonas plecoglossicida is a lethal pathogen of ayu (Plecoglossus altivelis) in Japan and is responsible for substantial economic costs to ayu culture. Previously, we demonstrated the efficacy of phage therapy against P. plecoglossicida infection using two lytic phages (PPpW-3 and PPpW-4) (S. C. Park, I. Shimamura, M. Fukunaga, K. Mori, and T. Nakai, Appl Environ Microbiol 66:1416-1422, 2000, http://dx.doi.org/10.1128/AEM.66.4.1416-1422.2000; S. C. Park and T. Nakai, Dis Aquat Org 53:33-39, 2003, http://dx.doi.org/10.3354/dao053033). In the present study, the complete genome sequences of these therapeutic P. plecoglossicida phages were determined and analyzed for deleterious factors as therapeutic agents. The genome of PPpW-3 (myovirus) consisted of 43,564 bp with a GC content of 61.1% and 66 predicted open reading frames (ORFs). Approximately half of the genes were similar to the genes of the Escherichia coli phage vB_EcoM_ECO1230-10 (myovirus). The genome of PPpW-4 (podovirus) consisted of 41,386 bp with a GC content of 56.8% and 50 predicted ORFs. More than 70% of the genes were similar to the genes of Pseudomonas fluorescens phage ϕIBB-PF7A and Pseudomonas putida phage ϕ15 (podoviruses). The whole-genome analysis revealed that no known virulence genes were present in PPpW-3 and PPpW-4. An integrase gene was found in PPpW-3, but other factors used for lysogeny were not confirmed. The PCR detection of phage genes in phage-resistant variants provided no evidence of lysogenic activity in PPpW-3 and PPpW-4. We conclude that these two lytic phages qualify as therapeutic agents. PMID:25416766

  9. Complete Genome Sequence Analysis of Two Pseudomonas plecoglossicida Phages, Potential Therapeutic Agents

    PubMed Central

    Yasuike, Motoshige; Nakamura, Yoji; Shigenobu, Yuya; Fujiwara, Atushi; Sano, Motohiko; Nakai, Toshihiro

    2014-01-01

    Pseudomonas plecoglossicida is a lethal pathogen of ayu (Plecoglossus altivelis) in Japan and is responsible for substantial economic costs to ayu culture. Previously, we demonstrated the efficacy of phage therapy against P. plecoglossicida infection using two lytic phages (PPpW-3 and PPpW-4) (S. C. Park, I. Shimamura, M. Fukunaga, K. Mori, and T. Nakai, Appl Environ Microbiol 66:1416–1422, 2000, http://dx.doi.org/10.1128/AEM.66.4.1416-1422.2000; S. C. Park and T. Nakai, Dis Aquat Org 53:33–39, 2003, http://dx.doi.org/10.3354/dao053033). In the present study, the complete genome sequences of these therapeutic P. plecoglossicida phages were determined and analyzed for deleterious factors as therapeutic agents. The genome of PPpW-3 (myovirus) consisted of 43,564 bp with a GC content of 61.1% and 66 predicted open reading frames (ORFs). Approximately half of the genes were similar to the genes of the Escherichia coli phage vB_EcoM_ECO1230-10 (myovirus). The genome of PPpW-4 (podovirus) consisted of 41,386 bp with a GC content of 56.8% and 50 predicted ORFs. More than 70% of the genes were similar to the genes of Pseudomonas fluorescens phage ϕIBB-PF7A and Pseudomonas putida phage ϕ15 (podoviruses). The whole-genome analysis revealed that no known virulence genes were present in PPpW-3 and PPpW-4. An integrase gene was found in PPpW-3, but other factors used for lysogeny were not confirmed. The PCR detection of phage genes in phage-resistant variants provided no evidence of lysogenic activity in PPpW-3 and PPpW-4. We conclude that these two lytic phages qualify as therapeutic agents. PMID:25416766

  10. A novel nuclear factor erythroid 2-related factor 2 (Nrf2) activator RS9 attenuates brain injury after ischemia reperfusion in mice.

    PubMed

    Yamauchi, Keita; Nakano, Yusuke; Imai, Takahiko; Takagi, Toshinori; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Iwama, Toru; Hara, Hideaki

    2016-10-01

    Recanalization of occluded vessels leads to ischemia-reperfusion injury (IRI), with oxidative stress as one of the main causes of injury, despite the fact that recanalization therapy is the most effective treatment for ischemic stroke. The nuclear factor erythroid 2-related factor 2 (Nrf2) is one of the transcription factors which has an essential role in protection against oxidative stress. RS9 is a novel Nrf2 activator obtained from bardoxolone methyl (BARD), an Nrf2 activator that has already been tested in a clinical trial, using a biotransformation technique. RS9 has been reported to lead to higher Nrf2 activation and less cytotoxicity than BARD. In this study, we investigated the effects of RS9 on IRI. Mice were intraperitoneally treated immediately after 2h of transient middle cerebral artery occlusion (MCAO) with a vehicle solution or 0.2mg/kg of RS9. Post-onset treatment of RS9 attenuated the infarct volume and improved neurological deficits 22h after reperfusion. RS9 activated Nrf2 2 and 6h after reperfusion and activated heme oxygenase-1 at 6 and 22h after reperfusion. RS9 also attenuated the phosphorylation of NF-κB p65 2 and 6h after reperfusion. Finally, RS9 improved the survival rate and neurological deficits 7days after MCAO. Our results suggest that the activation of Nrf2 by RS9 has a neuroprotective effect, mediated by attenuating both oxidative stress and neuroinflammation, and that RS9 is an effective therapeutic candidate for the treatment of IRI. PMID:27474227

  11. Following cell-fate in E. coli after infection by phage lambda.

    PubMed

    Zeng, Lanying; Golding, Ido

    2011-01-01

    : production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired. PMID:22025187

  12. Enhancing and initiating phage-based therapies

    PubMed Central

    Serwer, Philip; Wright, Elena T; Chang, Juan T; Liu, Xiangan

    2014-01-01

    Drug development has typically been a primary foundation of strategy for systematic, long-range management of pathogenic cells. However, drug development is limited in speed and flexibility when response is needed to changes in pathogenic cells, especially changes that produce drug-resistance. The high replication speed and high diversity of phages are potentially useful for increasing both response speed and response flexibility when changes occur in either drug resistance or other aspects of pathogenic cells. We present strategy, with some empirical details, for (1) using modern molecular biology and biophysics to access these advantages during the phage therapy of bacterial infections, and (2) initiating use of phage capsid-based drug delivery vehicles (DDVs) with procedures that potentially overcome both drug resistance and other present limitations in the use of DDVs for the therapy of neoplasms. The discussion of phage therapy includes (a) historical considerations, (b) changes that appear to be needed in clinical tests if use of phage therapy is to be expanded, (c) recent work on novel phages and its potential use for expanding the capabilities of phage therapy and (d) an outline for a strategy that encompasses both theory and practice for expanding the applications of phage therapy. The discussion of DDVs starts by reviewing current work on DDVs, including work on both liposomal and viral DDVs. The discussion concludes with some details of the potential use of permeability constrained phage capsids as DDVs. PMID:26713220

  13. Experimental Phage Therapy for Burkholderia pseudomallei Infection

    PubMed Central

    Leang-Chung, Choh; Vellasamy, Kumutha Malar; Mariappan, Vanitha; Li-Yen, Chang; Vadivelu, Jamuna

    2016-01-01

    Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen intrinsically resistant to a variety of antibiotics. Phages have been developed for use as an alternative treatment therapy, particularly for bacterial infections that do not respond to conventional antibiotics. In this study, we investigated the use of phages to treat cells infected with B. pseudomallei. Phage C34 isolated from seawater was purified and characterised on the basis of its host range and morphology using transmission electron microscopy (TEM). Phage C34 was able to lyse 39.5% of B. pseudomallei clinical strains. Due to the presence of contractile tail, phage C34 is classified as a member of the family Myoviridae, a tailed double-stranded DNA virus. When 2 × 105 A549 cells were exposed to 2 × 107 PFU of phage C34, 24 hours prior to infection with 2 × 106 CFU of B. pseudomallei, it was found that the survivability of the cells increased to 41.6 ± 6.8% as compared to 22.8 ± 6.0% in untreated control. Additionally, application of phage successfully rescued 33.3% of mice infected with B. pseudomallei and significantly reduced the bacterial load in the spleen of the phage-treated mice. These findings indicate that phage can be a potential antimicrobial agent for B. pseudomallei infections. PMID:27387381

  14. Systematic annotation and analysis of “virmugens” - virulence factors whose mutants can be used as live attenuated vaccines

    PubMed Central

    Racz, Rebecca; Chung, Monica; Xiang, Zuoshuang; He, Yongqun

    2012-01-01

    Live attenuated vaccines are usually generated by mutation of genes encoding virulence factors. “Virmugen” is coined here to represent a gene that encodes for a virulent factor of a pathogen and has been proven feasible in animal models to make a live attenuated vaccine by knocking out this gene. Not all virulence factors are virmugens. VirmugenDB is a web-based virmugen database (http://www.violinet.org/virmugendb). Currently, VirmugenDB includes 225 virmugens that have been verified to be valuable for vaccine development against 57 bacterial, viral, and protozoan pathogens. Bioinformatics analysis has revealed significant patterns in virmugens. For example, 10 Gram-negative and one Gram-positive bacterial aroA genes are virmugens. A sequence analysis has revealed at least 50% of identities in the protein sequences of the 10 Gram-negative bacterial aroA virmugens. As a pathogen case study, Brucella virmugens were analyzed. Out of 15 verified Brucella virmugens, six are related to carbohydrate or nucleotide transport and metabolism, and two involving cell membrane biogenesis. In addition, 54 virmugens from 24 viruses and 12 virmugens from 4 parasites are also stored in VirmugenDB. Virmugens tend to involve metabolism of nutrients (e.g., amino acids, carbohydrates, and nucleotides) and cell membrane formation. Host genes whose expressions were regulated by virmugen mutation vaccines or wild type virulent pathogens have also been annotated and systematically compared. The bioinformatics annotation and analysis of virmugens helps elucidate enriched virmugen profiles and the mechanisms of protective immunity, and further supports rational vaccine design. PMID:23219434

  15. Antagonism of Stem Cell Factor/c-kit Signaling Attenuates Neonatal Chronic Hypoxia-Induced Pulmonary Vascular Remodeling

    PubMed Central

    Young, Karen C; Torres, Eneida; Hehre, Dorothy; Wu, Shu; Suguihara, Cleide; Hare, Joshua M.

    2015-01-01

    Background Accumulating evidence suggests that c-kit positive cells are present in the remodeled pulmonary vasculature bed of patients with pulmonary hypertension (PH). Whether stem cell factor (SCF)/ c-kit regulated pathways potentiate pulmonary vascular remodeling is unknown. Here, we tested the hypothesis that attenuated c-kit signaling would decrease chronic hypoxia-induced pulmonary vascular remodeling by decreasing pulmonary vascular cell mitogenesis. Methods Neonatal FVB/NJ mice treated with non-immune IgG (PL), or c-kit neutralizing antibody (ACK2) as well as c-kit mutant mice (WBB6F1- Kit W− v/ +) and their congenic controls, were exposed to normoxia (FiO2=0.21) or hypoxia (FiO2=0.12) for two weeks. Following this exposure, right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH), pulmonary vascular cell proliferation and remodeling were evaluated. Results As compared to chronically hypoxic controls, c-kit mutant mice had decreased RVSP, RVH, pulmonary vascular remodeling and proliferation. Consistent with these findings, administration of ACK2 to neonatal mice with chronic hypoxia-induced PH decreased RVSP, RVH, pulmonary vascular cell proliferation and remodeling. This attenuation in PH was accompanied by decreased extracellular signal-regulated protein kinase (ERK) 1/2 activation. Conclusion SCF/c-kit signaling may potentiate chronic hypoxia-induced vascular remodeling by modulating ERK activation. Inhibition of c-kit activity may be a potential strategy to alleviate PH. PMID:26705118

  16. Rosmarinic Acid Attenuates Sodium Taurocholate-Induced Acute Pancreatitis in Rats by Inhibiting Nuclear Factor-κB Activation.

    PubMed

    Fan, Yu-Ting; Yin, Guo-Jian; Xiao, Wen-Qin; Qiu, Lei; Yu, Ge; Hu, Yan-Ling; Xing, Miao; Wu, De-Qing; Cang, Xiao-Feng; Wan, Rong; Wang, Xing-Peng; Hu, Guo-Yong

    2015-01-01

    Rosmarinic Acid (RA), a caffeic acid ester, has been shown to exert anti-inflammation, anti-oxidant and antiallergic effects. Our study aimed to investigate the effect of RA in sodium taurocholate ( NaTC )-induced acute pancreatitis, both in vivo and in vitro. In vivo, RA (50 mg/kg) was administered intraperitoneally 2 h before sodium taurocholate injection. Rats were sacrificed 12 h, 24 h or 48 h after sodium taurocholate injection. Pretreatment with RA significantly ameliorated pancreas histopathological changes, decreased amylase and lipase activities in serum, lowered myeloperoxidase activity in the pancreas, reduced systematic and pancreatic interleukin-1 β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) levels, and inhibited NF-κB translocation in pancreas. In vitro, pretreating the fresh rat pancreatic acinar cells with 80 μ mol/L RA 2 h before 3750 nmol/L sodium taurocholate or 10 ng/L TNF-α administration significantly attenuated the reduction of isolated pancreatic acinar cell viability and inhibited the nuclear activation and translocation of NF-κB. Based on our findings, RA appears to attenuate damage in sodium taurocholate-induced acute pancreatitis and reduce the release of inflammatory cytokines by inhibiting the activation of NF-κB. These findings might provide a basis for investigating the therapeutic role of RA in managing acute pancreatits. PMID:26364660

  17. Overexpression of hypoxia-inducible factor prolyl hydoxylase-2 attenuates hypoxia-induced vascular endothelial growth factor expression in luteal cells.

    PubMed

    Zhang, Zhenghong; Pang, Xunsheng; Tang, Zonghao; Yin, Dingzhong; Wang, Zhengchao

    2015-09-01

    Vascular endothelial growth factor (VEGF)-dependent angiogenesis has a crucial role in the corpus luteum formation and their functional maintenances in mammalian ovaries. A previous study by our group reported that activation of hypoxia‑inducible factor (HIF)‑1α signaling contributes to the regulation of VEGF expression in the luteal cells (LCs) in response to hypoxia and human chorionic gonadotropin. The present study was designed to test the hypothesis that HIF prolyl‑hydroxylases (PHDs) are expressed in LCs and overexpression of PHD2 attenuates the expression of VEGF induced by hypoxia in LCs. PHD2-overexpressing plasmid was transfected into LC2 cells, and successful plasmid transfection and expression was confirmed by reverse transcription quantitative polymerase chain reaction and western blot analysis. In addition, the present study investigated changes of HIF‑1α and VEGF expression after incubation under hypoxic conditions and PHD2 transfection. PHD2 expression was significantly higher expressed than the other two PHD isoforms, indicating its major role in LCs. Moreover, a significant increase of VEGF mRNA expression was identified after incubation under hypoxic conditions, which was, however, attenuated by PHD2 overexpression in LCs. Further analysis also indicated that this hypoxia‑induced increase in the mRNA expression of VEGF was consistent with increases in the protein levels of HIF‑1α, which is regulated by PHD-mediated degradation. In conclusion, the results of the present study indicated that PHD2 is the main PHD expressed in LCs and hypoxia‑induced VEGF expression can be attenuated by PHD2 overexpression through HIF‑1α‑mediated mechanisms in LCs. This PHD2-mediated transcriptional activation may be one of the mechanisms regulating VEGF expression in LCs during mammalian corpus luteum development. PMID:25975603

  18. Coordinate post-transcriptional repression of Dpp-dependent transcription factors attenuates signal range during development

    PubMed Central

    Newton, Fay G.; Harris, Robin E.; Sutcliffe, Catherine; Ashe, Hilary L.

    2015-01-01

    Precise control of the range of signalling molecule action is crucial for correct cell fate patterning during development. For example, Drosophila ovarian germline stem cells (GSCs) are maintained by exquisitely short-range BMP signalling from the niche. In the absence of BMP signalling, one GSC daughter differentiates into a cystoblast (CB) and this fate is stabilised by Brain tumour (Brat) and Pumilio (Pum)-mediated post-transcriptional repression of mRNAs, including that encoding the Dpp transducer, Mad. However, the identity of other repressed mRNAs and the mechanism of post-transcriptional repression are currently unknown. Here, we identify the Medea and schnurri mRNAs, which encode transcriptional regulators required for activation and/or repression of Dpp target genes, as additional Pum-Brat targets, suggesting that tripartite repression of the transducers is deployed to desensitise the CB to Dpp. In addition, we show that repression by Pum-Brat requires recruitment of the CCR4 and Pop2 deadenylases, with knockdown of deadenylases in vivo giving rise to ectopic GSCs. Consistent with this, Pum-Brat repression leads to poly(A) tail shortening and mRNA degradation in tissue culture cells, and we detect a reduced number of Mad and shn transcripts in the CB relative to the GSC based on single molecule mRNA quantitation. Finally, we show generality of the mechanism by demonstrating that Brat also attenuates pMad and Dpp signalling range in the early embryo. Together our data serve as a platform for understanding how post-transcriptional repression restricts interpretation of BMPs and other cell signals in order to allow robust cell fate patterning during development. PMID:26293305

  19. Chronic Tumor Necrosis Factor Alters T Cell Responses by Attenuating T Cell Receptor Signaling

    PubMed Central

    Cope, Andrew P.; Liblau, Roland S.; Yang, Xiao-Dong; Congia, Mauro; Laudanna, Carlo; Schreiber, Robert D.; Probert, Lesley; Kollias, George; McDevitt, Hugh O.

    1997-01-01

    Repeated injections of adult mice with recombinant murine TNF prolong the survival of NZB/W F1 mice, and suppress type I insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. To determine whether repeated TNF injections suppress T cell function in adult mice, we studied the responses of influenza hemagglutinin-specific T cells derived from T cell receptor (HNT-TCR) transgenic mice. Treatment of adult mice with murine TNF for 3 wk suppressed a broad range of T cell responses, including proliferation and cytokine production. Furthermore, T cell responses of HNT-TCR transgenic mice also expressing the human TNF-globin transgene were markedly reduced compared to HNT-TCR single transgenic littermates, indicating that sustained p55 TNF-R signaling is sufficient to suppress T cell function in vivo. Using a model of chronic TNF exposure in vitro, we demonstrate that (a) chronic TNF effects are dose and time dependent, (b) TNF suppresses the responses of both Th1 and Th2 T helper subsets, (c) the suppressive effects of endogenous TNF produced in T cell cultures could be reversed with neutralizing monoclonal antibodies to TNF, and (d) prolonged TNF exposure attenuates T cell receptor signaling. The finding that anti-TNF treatment in vivo enhances T cell proliferative responses and cytokine production provides evidence for a novel regulatory effect of TNF on T cells in healthy laboratory mice. These effects are more pronounced in chronic inflammatory disease. In addition, our data provide a mechanism through which prolonged TNF exposure suppresses disease in animal models of autoimmunity. PMID:9151895

  20. A virulent phage infecting Lactococcus garvieae, with homology to Lactococcus lactis phages.

    PubMed

    Eraclio, Giovanni; Tremblay, Denise M; Lacelle-Côté, Alexia; Labrie, Simon J; Fortina, Maria Grazia; Moineau, Sylvain

    2015-12-01

    A new virulent phage belonging to the Siphoviridae family and able to infect Lactococcus garvieae strains was isolated from compost soil. Phage GE1 has a prolate capsid (56 by 38 nm) and a long noncontractile tail (123 nm). It had a burst size of 139 and a latent period of 31 min. Its host range was limited to only two L. garvieae strains out of 73 tested. Phage GE1 has a double-stranded DNA genome of 24,847 bp containing 48 predicted open reading frames (ORFs). Putative functions could be assigned to only 14 ORFs, and significant matches in public databases were found for only 17 ORFs, indicating that GE1 is a novel phage and its genome contains several new viral genes and encodes several new viral proteins. Of these 17 ORFs, 16 were homologous to deduced proteins of virulent phages infecting the dairy bacterium Lactococcus lactis, including previously characterized prolate-headed phages. Comparative genome analysis confirmed the relatedness of L. garvieae phage GE1 to L. lactis phages c2 (22,172 bp) and Q54 (26,537 bp), although its genome organization was closer to that of phage c2. Phage GE1 did not infect any of the 58 L. lactis strains tested. This study suggests that phages infecting different lactococcal species may have a common ancestor. PMID:26407890

  1. A Virulent Phage Infecting Lactococcus garvieae, with Homology to Lactococcus lactis Phages

    PubMed Central

    Eraclio, Giovanni; Tremblay, Denise M.; Lacelle-Côté, Alexia; Labrie, Simon J.; Fortina, Maria Grazia

    2015-01-01

    A new virulent phage belonging to the Siphoviridae family and able to infect Lactococcus garvieae strains was isolated from compost soil. Phage GE1 has a prolate capsid (56 by 38 nm) and a long noncontractile tail (123 nm). It had a burst size of 139 and a latent period of 31 min. Its host range was limited to only two L. garvieae strains out of 73 tested. Phage GE1 has a double-stranded DNA genome of 24,847 bp containing 48 predicted open reading frames (ORFs). Putative functions could be assigned to only 14 ORFs, and significant matches in public databases were found for only 17 ORFs, indicating that GE1 is a novel phage and its genome contains several new viral genes and encodes several new viral proteins. Of these 17 ORFs, 16 were homologous to deduced proteins of virulent phages infecting the dairy bacterium Lactococcus lactis, including previously characterized prolate-headed phages. Comparative genome analysis confirmed the relatedness of L. garvieae phage GE1 to L. lactis phages c2 (22,172 bp) and Q54 (26,537 bp), although its genome organization was closer to that of phage c2. Phage GE1 did not infect any of the 58 L. lactis strains tested. This study suggests that phages infecting different lactococcal species may have a common ancestor. PMID:26407890

  2. A Novel Small-molecule Tumor Necrosis Factor α Inhibitor Attenuates Inflammation in a Hepatitis Mouse Model*

    PubMed Central

    Ma, Li; Gong, Haiyan; Zhu, Haiyan; Ji, Qing; Su, Pei; Liu, Peng; Cao, Shannan; Yao, Jianfeng; Jiang, Linlin; Han, Mingzhe; Ma, Xiaotong; Xiong, Dongsheng; Luo, Hongbo R.; Wang, Fei; Zhou, Jiaxi; Xu, Yuanfu

    2014-01-01

    Overexpression of tumor necrosis factor α (TNFα) is a hallmark of many inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and septic shock and hepatitis, making it a potential therapeutic target for clinical interventions. To explore chemical inhibitors against TNFα activity, we applied computer-aided drug design combined with in vitro and cell-based assays and identified a lead chemical compound, (E)-4-(2-(4-chloro-3-nitrophenyl) (named as C87 thereafter), which directly binds to TNFα, potently inhibits TNFα-induced cytotoxicity (IC50 = 8.73 μm) and effectively blocks TNFα-triggered signaling activities. Furthermore, by using a murine acute hepatitis model, we showed that C87 attenuates TNFα-induced inflammation, thereby markedly reducing injuries to the liver and improving animal survival. Thus, our results lead to a novel and highly specific small-molecule TNFα inhibitor, which can be potentially used to treat TNFα-mediated inflammatory diseases. PMID:24634219

  3. Mammalian Host-Versus-Phage immune response determines phage fate in vivo

    PubMed Central

    Hodyra-Stefaniak, Katarzyna; Miernikiewicz, Paulina; Drapała, Jarosław; Drab, Marek; Jończyk-Matysiak, Ewa; Lecion, Dorota; Kaźmierczak, Zuzanna; Beta, Weronika; Majewska, Joanna; Harhala, Marek; Bubak, Barbara; Kłopot, Anna; Górski, Andrzej; Dąbrowska, Krystyna

    2015-01-01

    Emerging bacterial antibiotic resistance draws attention to bacteriophages as a therapeutic alternative to treat bacterial infection. Examples of phage that combat bacteria abound. However, despite careful testing of antibacterial activity in vitro, failures nevertheless commonly occur. We investigated immunological response of phage antibacterial potency in vivo. Anti-phage activity of phagocytes, antibodies, and serum complement were identified by direct testing and by high-resolution fluorescent microscopy. We accommodated the experimental data into a mathematical model. We propose a universal schema of innate and adaptive immunity impact on phage pharmacokinetics, based on the results of our numerical simulations. We found that the mammalian-host response to infecting bacteria causes the concomitant removal of phage from the system. We propose the notion that this effect as an indirect pathway of phage inhibition by bacteria with significant relevance for the clinical outcome of phage therapy. PMID:26440922

  4. Kaiso depletion attenuates transforming growth factor-β signaling and metastatic activity of triple-negative breast cancer cells.

    PubMed

    Bassey-Archibong, B I; Kwiecien, J M; Milosavljevic, S B; Hallett, R M; Rayner, L G A; Erb, M J; Crawford-Brown, C J; Stephenson, K B; Bédard, P-A; Hassell, J A; Daniel, J M

    2016-01-01

    Triple-negative breast cancers (TNBCs) represent a subset of breast tumors that are highly aggressive and metastatic, and are responsible for a disproportionate number of breast cancer-related deaths. Several studies have postulated a role for the epithelial-to-mesenchymal transition (EMT) program in the increased aggressiveness and metastatic propensity of TNBCs. Although EMT is essential for early vertebrate development and wound healing, it is frequently co-opted by cancer cells during tumorigenesis. One prominent signaling pathway involved in EMT is the transforming growth factor-β (TGFβ) pathway. In this study, we report that the novel POZ-ZF transcription factor Kaiso is highly expressed in TNBCs and correlates with a shorter metastasis-free survival. Notably, Kaiso expression is induced by the TGFβ pathway and silencing Kaiso expression in the highly invasive breast cancer cell lines, MDA-MB-231 (hereafter MDA-231) and Hs578T, attenuated the expression of several EMT-associated proteins (Vimentin, Slug and ZEB1), abrogated TGFβ signaling and TGFβ-dependent EMT. Moreover, Kaiso depletion attenuated the metastasis of TNBC cells (MDA-231 and Hs578T) in a mouse model. Although high Kaiso and high TGFβR1 expression is associated with poor overall survival in breast cancer patients, overexpression of a kinase-active TGFβR1 in the Kaiso-depleted cells was insufficient to restore the metastatic potential of these cells, suggesting that Kaiso is a key downstream component of TGFβ-mediated pro-metastatic responses. Collectively, these findings suggest a critical role for Kaiso in TGFβ signaling and the metastasis of TNBCs. PMID:26999717

  5. Kaiso depletion attenuates transforming growth factor-β signaling and metastatic activity of triple-negative breast cancer cells

    PubMed Central

    Bassey-Archibong, B I; Kwiecien, J M; Milosavljevic, S B; Hallett, R M; Rayner, L G A; Erb, M J; Crawford-Brown, C J; Stephenson, K B; Bédard, P-A; Hassell, J A; Daniel, J M

    2016-01-01

    Triple-negative breast cancers (TNBCs) represent a subset of breast tumors that are highly aggressive and metastatic, and are responsible for a disproportionate number of breast cancer-related deaths. Several studies have postulated a role for the epithelial-to-mesenchymal transition (EMT) program in the increased aggressiveness and metastatic propensity of TNBCs. Although EMT is essential for early vertebrate development and wound healing, it is frequently co-opted by cancer cells during tumorigenesis. One prominent signaling pathway involved in EMT is the transforming growth factor-β (TGFβ) pathway. In this study, we report that the novel POZ-ZF transcription factor Kaiso is highly expressed in TNBCs and correlates with a shorter metastasis-free survival. Notably, Kaiso expression is induced by the TGFβ pathway and silencing Kaiso expression in the highly invasive breast cancer cell lines, MDA-MB-231 (hereafter MDA-231) and Hs578T, attenuated the expression of several EMT-associated proteins (Vimentin, Slug and ZEB1), abrogated TGFβ signaling and TGFβ-dependent EMT. Moreover, Kaiso depletion attenuated the metastasis of TNBC cells (MDA-231 and Hs578T) in a mouse model. Although high Kaiso and high TGFβR1 expression is associated with poor overall survival in breast cancer patients, overexpression of a kinase-active TGFβR1 in the Kaiso-depleted cells was insufficient to restore the metastatic potential of these cells, suggesting that Kaiso is a key downstream component of TGFβ-mediated pro-metastatic responses. Collectively, these findings suggest a critical role for Kaiso in TGFβ signaling and the metastasis of TNBCs. PMID:26999717

  6. Phage-displayed peptide libraries

    PubMed Central

    Zwick, Michael B; Shen, Juqun; Scott, Jamie K

    2014-01-01

    Over the past year, significant advances have been achieved through the use of phage-displayed peptide libraries. A wide variety of bioactive molecules, including antibodies, receptors and enzymes, have selected high-affinity and/or highly-specific peptide ligands from a number of different types of peptide library. The demonstrated therapeutic potential of some of these peptides, as well as new insights into protein structure and function that peptide ligands have provided, highlight the progress made within this rapidly-expanding field. PMID:9720267

  7. CRISPR Immunity Drives Rapid Phage Genome Evolution in Streptococcus thermophilus

    PubMed Central

    Paez-Espino, David; Sharon, Itai; Morovic, Wesley; Stahl, Buffy; Thomas, Brian C.

    2015-01-01

    ABSTRACT Many bacteria rely on CRISPR-Cas systems to provide adaptive immunity against phages, predation by which can shape the ecology and functioning of microbial communities. To characterize the impact of CRISPR immunization on phage genome evolution, we performed long-term bacterium-phage (Streptococcus thermophilus-phage 2972) coevolution experiments. We found that in this species, CRISPR immunity drives fixation of single nucleotide polymorphisms that accumulate exclusively in phage genome regions targeted by CRISPR. Mutation rates in phage genomes highly exceed those of the host. The presence of multiple phages increased phage persistence by enabling recombination-based formation of chimeric phage genomes in which sequences heavily targeted by CRISPR were replaced. Collectively, our results establish CRISPR-Cas adaptive immunity as a key driver of phage genome evolution under the conditions studied and highlight the importance of multiple coexisting phages for persistence in natural systems. PMID:25900652

  8. Inhibition of hypoxia inducible factor-1α attenuates abdominal aortic aneurysm progression through the down-regulation of matrix metalloproteinases.

    PubMed

    Tsai, Shih-Hung; Huang, Po-Hsun; Hsu, Yu-Juei; Peng, Yi-Jen; Lee, Chien-Hsing; Wang, Jen-Chun; Chen, Jaw-Wen; Lin, Shing-Jong

    2016-01-01

    Hypoxia inducible factor-1α (HIF-1α) pathway is associated with many vascular diseases, including atherosclerosis, arterial aneurysms, pulmonary hypertension and chronic venous diseases. Significant HIF-1α expression could be found at the rupture edge at human abdominal aortic aneurysm (AAA) tissues. While our initial in vitro experiments had shown that deferoxamine (DFO) could attenuate angiotensin II (AngII) induced endothelial activations; we unexpectedly found that DFO augmented the severity of AngII-induced AAA, at least partly through increased accumulation of HIF-1α. The findings promoted us to test whether aneurysmal prone factors could up-regulate the expression of MMP-2 and MMP-9 through aberrantly increased HIF-1α and promote AAA development. AngII induced AAA in hyperlipidemic mice model was used. DFO, as a prolyl hydroxylase inhibitor, stabilized HIF-1α and augmented MMPs activities. Aneurysmal-prone factors induced HIF-1α can cause overexpression of MMP-2 and MMP-9 and promote aneurysmal progression. Pharmacological HIF-1α inhibitors, digoxin and 2-ME could ameliorate AngII induced AAA in vivo. HIF-1α is pivotal for the development of AAA. Our study provides a rationale for using HIF-1α inhibitors as an adjunctive medical therapy in addition to current cardiovascular risk-reducing regimens. PMID:27363580

  9. Inhibition of hypoxia inducible factor-1α attenuates abdominal aortic aneurysm progression through the down-regulation of matrix metalloproteinases

    PubMed Central

    Tsai, Shih-Hung; Huang, Po-Hsun; Hsu, Yu-Juei; Peng, Yi-Jen; Lee, Chien-Hsing; Wang, Jen-Chun; Chen, Jaw-Wen; Lin, Shing-Jong

    2016-01-01

    Hypoxia inducible factor-1α (HIF-1α) pathway is associated with many vascular diseases, including atherosclerosis, arterial aneurysms, pulmonary hypertension and chronic venous diseases. Significant HIF-1α expression could be found at the rupture edge at human abdominal aortic aneurysm (AAA) tissues. While our initial in vitro experiments had shown that deferoxamine (DFO) could attenuate angiotensin II (AngII) induced endothelial activations; we unexpectedly found that DFO augmented the severity of AngII-induced AAA, at least partly through increased accumulation of HIF-1α. The findings promoted us to test whether aneurysmal prone factors could up-regulate the expression of MMP-2 and MMP-9 through aberrantly increased HIF-1α and promote AAA development. AngII induced AAA in hyperlipidemic mice model was used. DFO, as a prolyl hydroxylase inhibitor, stabilized HIF-1α and augmented MMPs activities. Aneurysmal-prone factors induced HIF-1α can cause overexpression of MMP-2 and MMP-9 and promote aneurysmal progression. Pharmacological HIF-1α inhibitors, digoxin and 2-ME could ameliorate AngII induced AAA in vivo. HIF-1α is pivotal for the development of AAA. Our study provides a rationale for using HIF-1α inhibitors as an adjunctive medical therapy in addition to current cardiovascular risk-reducing regimens. PMID:27363580

  10. Hydrogen Sulfide Levels and Nuclear Factor-Erythroid 2-Related Factor 2 (NRF2) Activity Are Attenuated in the Setting of Critical Limb Ischemia (CLI)

    PubMed Central

    Islam, Kazi N; Polhemus, David J; Donnarumma, Erminia; Brewster, Luke P; Lefer, David J

    2015-01-01

    Background Cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase are endogenous enzymatic sources of hydrogen sulfide (H2S). Functions of H2S are mediated by several targets including ion channels and signaling proteins. Nuclear factor-erythroid 2-related factor 2 is responsible for the expression of antioxidant response element–regulated genes and is known to be upregulated by H2S. We examined the levels of H2S, H2S-producing enzymes, and nuclear factor-erythroid 2-related factor 2 activation status in skeletal muscle obtained from critical limb ischemia (CLI) patients. Methods and Results Gastrocnemius tissues were attained postamputation from human CLI and healthy control patients. We found mRNA and protein levels of cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase were significantly decreased in skeletal muscle of CLI patients as compared to control. H2S and sulfane sulfur levels were significantly decreased in skeletal muscle of CLI patients. We also observed significant reductions in nuclear factor-erythroid 2-related factor 2 activation as well as antioxidant proteins, such as Cu, Zn-superoxide dismutase, catalase, and glutathione peroxidase in skeletal muscle of CLI patients. Biomarkers of oxidative stress, such as malondialdehyde and protein carbonyl formation, were significantly increased in skeletal muscle of CLI patients as compared to healthy controls. Conclusions The data demonstrate that H2S bioavailability and nuclear factor-erythroid 2-related factor 2 activation are both attenuated in CLI tissues concomitant with significantly increased oxidative stress. Reductions in the activity of H2S-producing enzymes may contribute to the pathogenesis of CLI. PMID:25977470

  11. Tumor necrosis factor-α inhibition attenuates middle cerebral artery remodeling but increases cerebral ischemic damage in hypertensive rats

    PubMed Central

    Girgla, Saavia S.; Moreno, Guillermo; McClain, Jonathon L.; Dorrance, Anne M.

    2014-01-01

    Hypertension causes vascular inflammation evidenced by an increase in perivascular macrophages and proinflammatory cytokines in the arterial wall. Perivascular macrophage depletion reduced tumor necrosis factor (TNF)-α expression in cerebral arteries of hypertensive rats and attenuated inward remodeling, suggesting that TNF-α might play a role in the remodeling process. We hypothesized that TNF-α inhibition would improve middle cerebral artery (MCA) structure and reduce damage after cerebral ischemia in hypertensive rats. Six-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were treated with the TNF-α inhibitor etanercept (ETN; 1.25 mg·kg−1·day−1 ip daily) or PBS (equivolume) for 6 wk. The myogenic tone generation, postischemic dilation, and passive structure of MCAs were assessed by pressure myography. Cerebral ischemia was induced by MCA occlusion (MCAO). Myogenic tone was unchanged, but MCAs from SHRSP + ETN had larger passive lumen diameter and reduced wall thickness and wall-to-lumen ratio. Cerebral infarct size was increased in SHRSP + ETN after transient MCAO, despite an improvement in dilation of nonischemic MCA. The increase in infarct size was linked to a reduction in the number of microglia in the infarct core and upregulation of markers of classical macrophage/microglia polarization. There was no difference in infarct size after permanent MCAO or when untreated SHRSP subjected to transient MCAO were given ETN at reperfusion. Our data suggests that TNF-α inhibition attenuates hypertensive MCA remodeling but exacerbates cerebral damage following ischemia/reperfusion injury likely due to inhibition of the innate immune response of the brain. PMID:25015967

  12. Tumour necrosis factor receptors and apoptosis of alveolar macrophages during early infection with attenuated and virulent Mycobacterium bovis

    PubMed Central

    Rodrigues, Michele F; Alves, Caio C S; Figueiredo, Bárbara B M; Rezende, Alice B; Wohlres-Viana, Sabine; da Silva, Vânia Lúcia; Machado, Marco Antônio; Teixeira, Henrique C

    2013-01-01

    Apoptosis of macrophages has been reported as an effective host strategy to control the growth of intracellular pathogens, including pathogenic mycobacteria. Tumour necrosis factor-α (TNF-α) plays an important role in the modulation of apoptosis of infected macrophages. It exerts its biological activities via two distinct cell surface receptors, TNFR1 and TNFR2, whose extracellular domain can be released by proteolysis forming soluble TNF receptors (sTNFR1 and sTNFR2). The signalling through TNFR1 initiates the majority of the biological functions of TNF-α, leading to either cell death or survival whereas TNFR2 mediates primarily survival signals. Here, the expression of TNF-α receptors and the apoptosis of alveolar macrophages were investigated during the early phase of infection with attenuated and virulent mycobacteria in mice. A significant increase of apoptosis and high expression of TNFR1 were observed in alveolar macrophages at 3 and 7 days after infection with attenuated Mycobacterium bovis but only on day 7 in infection with the virulent M. bovis. Low surface expression of TNFR1 and increased levels of sTNFR1 on day 3 after infection by the virulent strain were associated with reduced rates of apoptotic macrophages. In addition, a significant reduction in apoptosis of alveolar macrophages was observed in TNFR1−/− mice at day 3 after bacillus Calmette–Guérin infection. These results suggest a potential role for TNFR1 in mycobacteria-induced alveolar macrophage apoptosis in vivo. In this scenario, shedding of TNFR1 seems to contribute to the modulation of macrophage apoptosis in a strain-dependent manner. PMID:23489296

  13. Diosgenin attenuates hepatic stellate cell activation through transforming growth factor-β/Smad signaling pathway

    PubMed Central

    Xie, Wei-Lin; Jiang, Rong; Shen, Xiao-Lu; Chen, Zhi-Yu; Deng, Xiao-Ming

    2015-01-01

    Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-β1 (TGF-β1) is considered to be the main stimuli factor responsible for the activation of HSC. Diosgenin is a steroidal saponin found in several plants including Solanum and Dioscorea species, and it inhibited high glucose-induced renal tubular fibrosis. However, the effects of diosgenin against hepatic fibrosis remain elusive. Therefore, in this study, we investigated the effects of diosgenin on TGF-β1-induced HSCs and elucidate the possible mechanism of its anti-fibrotic effect. Our results demonstrated that diosgenin inhibited TGF-β1-induced HSC proliferation, reduced the expression of collagen I and α-smooth muscle actin (α-SMA), as well as the expression of TGF-β receptor I (TGF-β RI) and II. Moreover, diosgenin suppressed TGF-β1-induced phosphorylation of Smad3 in HSCs. In conclusion, our data demonstrate that diosgenin inhibited HSC-T6 cell proliferation and activation, at least in part, via the TGF-β1/Smad signaling pathway. These results provide that diosgenin may have potential to treat liver fibrosis. PMID:26884947

  14. Diosgenin attenuates hepatic stellate cell activation through transforming growth factor-β/Smad signaling pathway.

    PubMed

    Xie, Wei-Lin; Jiang, Rong; Shen, Xiao-Lu; Chen, Zhi-Yu; Deng, Xiao-Ming

    2015-01-01

    Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-β1 (TGF-β1) is considered to be the main stimuli factor responsible for the activation of HSC. Diosgenin is a steroidal saponin found in several plants including Solanum and Dioscorea species, and it inhibited high glucose-induced renal tubular fibrosis. However, the effects of diosgenin against hepatic fibrosis remain elusive. Therefore, in this study, we investigated the effects of diosgenin on TGF-β1-induced HSCs and elucidate the possible mechanism of its anti-fibrotic effect. Our results demonstrated that diosgenin inhibited TGF-β1-induced HSC proliferation, reduced the expression of collagen I and α-smooth muscle actin (α-SMA), as well as the expression of TGF-β receptor I (TGF-β RI) and II. Moreover, diosgenin suppressed TGF-β1-induced phosphorylation of Smad3 in HSCs. In conclusion, our data demonstrate that diosgenin inhibited HSC-T6 cell proliferation and activation, at least in part, via the TGF-β1/Smad signaling pathway. These results provide that diosgenin may have potential to treat liver fibrosis. PMID:26884947

  15. Reduction of Nup107 attenuates the growth factor signaling in the senescent cells

    SciTech Connect

    Kim, Sung Young; Kang, Hyun Tae; Choi, Hae Ri; Park, Sang Chul

    2010-10-08

    Research highlights: {yields} Decreased expression of Nup107 in aged cells and organs. {yields} Depletion of Nup107 results in impaired nuclear translocation of p-ERK. {yields} Depletion of Nup107 affects downstream effectors of ERK signaling. {yields} Depletion of Nup107 inhibits cell proliferation of oligodendroglioma cells. -- Abstract: Hypo-responsiveness to growth factors is a fundamental feature of cellular senescence. In this study, we found markedly decreased level of Nup107, a key scaffold protein in nuclear pore complex assembly, in senescent human diploid fibroblasts as well as in organs of aged mice. Depletion of Nup107 by specific siRNA in young human diploid fibroblasts prevented the effective nuclear translocation of phosphorylated extracellular signal-regulated kinase (ERK) following epidermal growth factor (EGF) stimulation, and decreased the expression of c-Fos in consequence. The disturbances in ERK signaling in Nup107 depleted cells closely mirror the similar changes in senescent cells. Knockdown of Nup107 in anaplastic oligodendroglioma cells caused cell death, rather than growth retardation, indicating a greater sensitivity to Nup107 depletion in cancer cells than in normal cells. These findings support the notion that Nup107 may contribute significantly to the regulation of cell fate in aged and transformed cells by modulating nuclear trafficking of signal molecules.

  16. Targeting membrane proteins for antibody discovery using phage display.

    PubMed

    Jones, Martina L; Alfaleh, Mohamed A; Kumble, Sumukh; Zhang, Shuo; Osborne, Geoffrey W; Yeh, Michael; Arora, Neetika; Hou, Jeff Jia Cheng; Howard, Christopher B; Chin, David Y; Mahler, Stephen M

    2016-01-01

    A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b. PMID:27189586

  17. Targeting membrane proteins for antibody discovery using phage display

    PubMed Central

    Jones, Martina L.; Alfaleh, Mohamed A.; Kumble, Sumukh; Zhang, Shuo; Osborne, Geoffrey W.; Yeh, Michael; Arora, Neetika; Hou, Jeff Jia Cheng; Howard, Christopher B.; Chin, David Y.; Mahler, Stephen M.

    2016-01-01

    A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b. PMID:27189586

  18. Clear Plaque Mutants of Lactococcal Phage TP901-1

    PubMed Central

    Kot, Witold; Kilstrup, Mogens; Vogensen, Finn K.; Hammer, Karin

    2016-01-01

    We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome resequencing. Four of the mutants had different mutations in structural genes with no relation to the genetic switch. However all 8 mutants had a mutation in the cI repressor gene region. Three of these were located in the promoter and Shine-Dalgarno sequences and five in the N-terminal part of the encoded CI protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1. PMID:27258092

  19. Deletion of znuA virulence factor attenuates Brucella abortus and confers protection against wild-type challenge.

    PubMed

    Yang, Xinghong; Becker, Todd; Walters, Nancy; Pascual, David W

    2006-07-01

    znuA is known to be an important factor for survival and normal growth under low Zn(2+) concentrations for Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida. We hypothesized that the znuA gene present in Brucella melitensis 16 M would be similar to znuA in B. abortus and questioned whether it may also be an important factor for growth and virulence of Brucella abortus. Using the B. melitensis 16 M genome sequence, primers were designed to construct a B. abortus deletion mutant. A znuA knockout mutation in B. abortus 2308 (DeltaznuA) was constructed and found to be lethal in low-Zn(2+) medium. When used to infect macrophages, DeltaznuA B. abortus showed minimal growth. Further study with DeltaznuA B. abortus showed that its virulence in BALB/c mice was attenuated, and most of the bacteria were cleared from the spleen within 8 weeks. Protection studies confirmed the DeltaznuA mutant as a potential live vaccine, since protection against wild-type B. abortus 2308 challenge was as effective as that obtained with the RB51 or S19 vaccine strain. PMID:16790759

  20. The spliceosome assembly factor GEMIN2 attenuates the effects of temperature on alternative splicing and circadian rhythms

    PubMed Central

    Schlaen, Rubén Gustavo; Mancini, Estefanía; Sanchez, Sabrina Elena; Perez-Santángelo, Soledad; Rugnone, Matías L.; Simpson, Craig G.; Brown, John W. S.; Zhang, Xu; Chernomoretz, Ariel; Yanovsky, Marcelo J.

    2015-01-01

    The mechanisms by which poikilothermic organisms ensure that biological processes are robust to temperature changes are largely unknown. Temperature compensation, the ability of circadian rhythms to maintain a relatively constant period over the broad range of temperatures resulting from seasonal fluctuations in environmental conditions, is a defining property of circadian networks. Temperature affects the alternative splicing (AS) of several clock genes in fungi, plants, and flies, but the splicing factors that modulate these effects to ensure clock accuracy throughout the year remain to be identified. Here we show that GEMIN2, a spliceosomal small nuclear ribonucleoprotein assembly factor conserved from yeast to humans, modulates low temperature effects on a large subset of pre-mRNA splicing events. In particular, GEMIN2 controls the AS of several clock genes and attenuates the effects of temperature on the circadian period in Arabidopsis thaliana. We conclude that GEMIN2 is a key component of a posttranscriptional regulatory mechanism that ensures the appropriate acclimation of plants to daily and seasonal changes in temperature conditions. PMID:26170331

  1. BaeSR, involved in envelope stress response, protects against lysogenic conversion by Shiga toxin 2-encoding phages.

    PubMed

    Imamovic, Lejla; Martínez-Castillo, Alexandre; Benavides, Carmen; Muniesa, Maite

    2015-04-01

    Infection and lysogenic conversion with Shiga toxin-encoding bacteriophages (Stx phages) drive the emergence of new Shiga toxin-producing Escherichia coli strains. Phage attachment to the bacterial surface is the first stage of phage infection. Envelope perturbation causes activation of envelope stress responses in bacterial cells. Although many external factors are known to activate envelope stress responses, the role of these responses in the phage-bacterium interaction remains unexplored. Here, we investigate the link between three envelope signaling systems in E. coli (RcsBC, CpxAR, and BaeSR) and Stx2 phage infection by determining the success of bacterial lysogenic conversion. For this purpose, E. coli DH5α wild-type (WT) and mutant strains lacking RcsBC, CpxAR, or BaeSR signaling systems were incubated with a recombinant Stx2 phage (933W). Notably, the number of lysogens obtained with the BaeSR mutant was 5 log10 units higher than with the WT, and the same differences were observed when using 7 different Stx2 phages. To assess whether the membrane receptor used by Stx phages, BamA, was involved in the differences observed, bamA gene expression was monitored by reverse transcription-quantitative PCR (RT-qPCR) in all host strains. A 4-fold-higher bamA expression level was observed in the BaeSR mutant than in the WT strain, suggesting that differential expression of the receptor used by Stx phages accounted for the increase in the number of lysogenization events. Establishing the link between the role of stress responses and phage infection has important implications for understanding the factors affecting lysogenic conversion, which drives the emergence of new pathogenic clones. PMID:25624356

  2. Gonadotropin Surge-inhibiting/attenuating Factors: A Review of Current Evidence, Potential Applications, and Future Directions for Research

    PubMed Central

    VEGA, MARIO G.; ZAREK, SHVETHA M.; BHAGWAT, MEDHA; SEGARS, JAMES H.

    2016-01-01

    SUMMARY Animal studies in the 1980s suggested the existence of an ovarian hormone, termed gonadotropin surge-inhibiting/attenuating factor (GnSIF/AF), that modulates pituitary secretion of luteinizing hormone (LH). Given the importance of identifying regulatory factors of the hypothalamic-pituitary-ovarian axis and the accumulating data suggesting its existence, we conducted a comprehensive literature search using PubMed, Web of Science, Scopus, and Embase to identify articles related to GnSIF/AF. The search generated 161 publications, of which 97 were included in this study. Several attempts have been made to identify and characterize this hormone and several candidates have been identified, but the protein sequences of these putative GnSIF/AF factors differ widely from one study to another. In addition, while the RF-amide RFRP-3 is known foremost as a neuropeptide, some research supports an ovarian origin for this non-steroidal hormone, thereby suggesting a role for RFRP-3 either as a co-modulator of GnSIF/AF or as a gonadotropin-inhibiting factor in the hypothalamus (GnIH). Discovery of the KNDy neurons that modulate GnRH secretion, on the other hand, further encourages the search for substance(s) that modulate their activity and that indirectly affect LH secretion and the hypothalamic-pituitary-ovarian axis. While it has remained an elusive hormone, GnSIF/AF holds many potential applications for contraception, in vitro fertilization, and/or cancer as well as for understanding polycystic ovary syndrome, metabolic diseases, and/or pubertal development. In this review, we rigorously examine the available evidence regarding the existence of GnSIF/AF, previous attempts at its identification, limitations to its discovery, future directions of research, and potential clinical applications. PMID:25581424

  3. Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

    PubMed Central

    Gao, Run-Ping; Brigstock, David R

    2009-01-01

    AIM: To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2) on human hepatic stellate cell (HSC) function. METHODS: CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor (TGF)-β1 to the culture medium. Semi-quantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen I, while protein levels of each molecule in cell lysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry. RESULTS: In pTriEx2-transfected LX-2 cells, TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagen I, and an increase in produced and secreted CCN2 or extracellular collagen I protein levels. pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen I protein. Furthermore, the TGF-β1-induced increase in mRNA or protein for CCN2 or collagen I was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase. CONCLUSION: Endogenous CCN2 is a mediator of basal or TGF-β1-induced collagen I production in human HSCs and regulates entry of the cells into S phase. PMID:19673024

  4. Inactivation and reactivation of B. megatherium phage.

    PubMed

    NORTHROP, J H

    1955-11-20

    Preparation of Reversibly Inactivated (R.I.) Phage.- If B. megatherium phage (of any type, or in any stage of purification) is suspended in dilute salt solutions at pH 5-6, it is completely inactivated; i.e., it does not form plaques, or give rise to more phage when mixed with a sensitive organism (Northrop, 1954). The inactivation occurs when the phage is added to the dilute salt solution. If a suspension of the inactive phage in pH 7 peptone is titrated to pH 5 and allowed to stand, the activity gradually returns. The inactivation is therefore reversible. Properties of R.I. Phage.- The R.I. phage is adsorbed by sensitive cells at about the same rate as the active phage. It kills the cells, but no active phage is produced. The R.I. phage therefore has the properties of phage "ghosts" (Herriott, 1951) or of colicines (Gratia, 1925), or phage inactivated by ultraviolet light (Luria, 1947). The R.I. phage is sedimented in the centrifuge at the same rate as active phage. It is therefore about the same size as the active phage. The R.I. phage is most stable in pH 7, 5 per cent peptone, and may be kept in this solution for weeks at 0 degrees C. The rate of digestion of R.I. phage by trypsin, chymotrypsin, or desoxyribonuclease is about the same as that of active phage (Northrop, 1955 a). Effect of Various Substances on the Formation of R.I. Phage.- There is an equilibrium between R.I. phage and active phage. The R.I. form is the stable one in dilute salt solution, pH 5 to 6.5 and at low temperature (<20 degrees C.). At pH >6.5, in dilute salt solution, the R.I. phage changes to the active form. The cycle, active right harpoon over left harpoon inactive phage, may be repeated many times at 0 degrees C. by changing the pH of the solution back and forth between pH 7 and pH 6. Irreversible inactivation is caused by distilled water, some heavy metals, concentrated urea or quanidine solutions, and by l-arginine. Reversible inactivation is prevented by all salts tested (except

  5. Reporter Phage and Breath Tests: Emerging Phenotypic Assays for Diagnosing Active Tuberculosis, Antibiotic Resistance, and Treatment Efficacy

    PubMed Central

    Jain, Paras; Thaler, David S.; Maiga, Mamoudou; Timmins, Graham S.; Bishai, William R.; Hatfull, Graham F.; Larsen, Michelle H.; Jacobs, William R.

    2011-01-01

    The rapid and accurate diagnosis of active tuberculosis (TB) and its drug susceptibility remain a challenge. Phenotypic assays allow determination of antibiotic susceptibilities even if sequence data are not available or informative. We review 2 emerging diagnostic approaches, reporter phage and breath tests, both of which assay mycobacterial metabolism. The reporter phage signal, Green fluorescent protein (GFP) or β-galactosidase, indicates transcription and translation inside the recipient bacilli and its attenuation by antibiotics. Different breath tests assay, (1) exhaled antigen 85, (2) mycobacterial urease activity, and (3) detection by trained rats of disease-specific odor in sputum, have also been developed. When compared with culture, reporter phage assays shorten the time for initial diagnosis of drug susceptibility by several days. Both reporter phage and breath tests have promise as early markers to determine the efficacy of treatment. While sputum often remains smear and Mycobacterium tuberculosis DNA positive early in the course of efficacious antituberculous treatment, we predict that both breath and phage tests will rapidly become negative. If this hypothesis proves correct, phage assays and breath tests could become important surrogate markers in early bactericidal activity (EBA) studies of new antibiotics. PMID:21996696

  6. Evidence of Geobacter-associated phage in a uranium-contaminated aquifer

    PubMed Central

    Holmes, Dawn E; Giloteaux, Ludovic; Chaurasia, Akhilesh K; Williams, Kenneth H; Luef, Birgit; Wilkins, Michael J; Wrighton, Kelly C; Thompson, Courtney A; Comolli, Luis R; Lovley, Derek R

    2015-01-01

    Geobacter species may be important agents in the bioremediation of organic and metal contaminants in the subsurface, but as yet unknown factors limit the in situ growth of subsurface Geobacter well below rates predicted by analysis of gene expression or in silico metabolic modeling. Analysis of the genomes of five different Geobacter species recovered from contaminated subsurface sites indicated that each of the isolates had been infected with phage. Geobacter-associated phage sequences were also detected by metagenomic and proteomic analysis of samples from a uranium-contaminated aquifer undergoing in situ bioremediation, and phage particles were detected by microscopic analysis in groundwater collected from sediment enrichment cultures. Transcript abundance for genes from the Geobacter-associated phage structural proteins, tail tube Gp19 and baseplate J, increased in the groundwater in response to the growth of Geobacter species when acetate was added, and then declined as the number of Geobacter decreased. Western blot analysis of a Geobacter-associated tail tube protein Gp19 in the groundwater demonstrated that its abundance tracked with the abundance of Geobacter species. These results suggest that the enhanced growth of Geobacter species in the subsurface associated with in situ uranium bioremediation increased the abundance and activity of Geobacter-associated phage and show that future studies should focus on how these phages might be influencing the ecology of this site. PMID:25083935

  7. Evidence of Geobacter-associated phage in a uranium-contaminated aquifer.

    PubMed

    Holmes, Dawn E; Giloteaux, Ludovic; Chaurasia, Akhilesh K; Williams, Kenneth H; Luef, Birgit; Wilkins, Michael J; Wrighton, Kelly C; Thompson, Courtney A; Comolli, Luis R; Lovley, Derek R

    2015-02-01

    Geobacter species may be important agents in the bioremediation of organic and metal contaminants in the subsurface, but as yet unknown factors limit the in situ growth of subsurface Geobacter well below rates predicted by analysis of gene expression or in silico metabolic modeling. Analysis of the genomes of five different Geobacter species recovered from contaminated subsurface sites indicated that each of the isolates had been infected with phage. Geobacter-associated phage sequences were also detected by metagenomic and proteomic analysis of samples from a uranium-contaminated aquifer undergoing in situ bioremediation, and phage particles were detected by microscopic analysis in groundwater collected from sediment enrichment cultures. Transcript abundance for genes from the Geobacter-associated phage structural proteins, tail tube Gp19 and baseplate J, increased in the groundwater in response to the growth of Geobacter species when acetate was added, and then declined as the number of Geobacter decreased. Western blot analysis of a Geobacter-associated tail tube protein Gp19 in the groundwater demonstrated that its abundance tracked with the abundance of Geobacter species. These results suggest that the enhanced growth of Geobacter species in the subsurface associated with in situ uranium bioremediation increased the abundance and activity of Geobacter-associated phage and show that future studies should focus on how these phages might be influencing the ecology of this site. PMID:25083935

  8. Antirepression system associated with the life cycle switch in the temperate podoviridae phage SPC32H.

    PubMed

    Kim, Minsik; Ryu, Sangryeol

    2013-11-01

    Prophages switch from lysogenic to lytic mode in response to the host SOS response. The primary factor that governs this switch is a phage repressor, which is typically a host RecA-dependent autocleavable protein. Here, in an effort to reveal the mechanism underlying the phenotypic differences between the Salmonella temperate phages SPC32H and SPC32N, whose genome sequences differ by only two nucleotides, we identified a new class of Podoviridae phage lytic switch antirepressor that is structurally distinct from the previously reported Sipho- and Myoviridae phage antirepressors. The SPC32H repressor (Rep) is not cleaved by the SOS response but instead is inactivated by a small antirepressor (Ant), the expression of which is negatively controlled by host LexA. A single nucleotide mutation in the consensus sequence of the LexA-binding site, which overlaps with the ant promoter, results in constitutive Ant synthesis and consequently induces SPC32N to enter the lytic cycle. Numerous potential Ant homologues were identified in a variety of putative prophages and temperate Podoviridae phages, indicating that antirepressors may be widespread among temperate phages in the order Caudovirales to mediate a prudent prophage induction. PMID:23986584

  9. Antirepression System Associated with the Life Cycle Switch in the Temperate Podoviridae Phage SPC32H

    PubMed Central

    Kim, Minsik

    2013-01-01

    Prophages switch from lysogenic to lytic mode in response to the host SOS response. The primary factor that governs this switch is a phage repressor, which is typically a host RecA-dependent autocleavable protein. Here, in an effort to reveal the mechanism underlying the phenotypic differences between the Salmonella temperate phages SPC32H and SPC32N, whose genome sequences differ by only two nucleotides, we identified a new class of Podoviridae phage lytic switch antirepressor that is structurally distinct from the previously reported Sipho- and Myoviridae phage antirepressors. The SPC32H repressor (Rep) is not cleaved by the SOS response but instead is inactivated by a small antirepressor (Ant), the expression of which is negatively controlled by host LexA. A single nucleotide mutation in the consensus sequence of the LexA-binding site, which overlaps with the ant promoter, results in constitutive Ant synthesis and consequently induces SPC32N to enter the lytic cycle. Numerous potential Ant homologues were identified in a variety of putative prophages and temperate Podoviridae phages, indicating that antirepressors may be widespread among temperate phages in the order Caudovirales to mediate a prudent prophage induction. PMID:23986584

  10. Multipronged attenuation of macrophage-colony stimulating factor signaling by Epstein-Barr virus BARF1

    SciTech Connect

    Shim, Ann Hye-Ryong; Chang, Rhoda Ahn; Chen, Xiaoyan; Longnecker, Richard; He, Xiaolin

    2014-10-02

    The ubiquitous EBV causes infectious mononucleosis and is associated with several types of cancers. The EBV genome encodes an early gene product, BARF1, which contributes to pathogenesis, potentially through growth-altering and immune-modulating activities, but the mechanisms for such activities are poorly understood. We have determined the crystal structure of BARF1 in complex with human macrophage-colony stimulating factor (M-CSF), a hematopoietic cytokine with pleiotropic functions in development and immune response. BARF1 and M-CSF form a high-affinity, stable, ring-like complex in both solution and the crystal, with a BARF1 hexameric ring surrounded by three M-CSF dimers in triangular array. The binding of BARF1 to M-CSF dramatically reduces but does not completely abolish M-CSF binding and signaling through its cognate receptor FMS. A three-pronged down-regulation mechanism is proposed to explain the biological effect of BARF1 on M-CSF:FMS signaling. These prongs entail control of the circulating and effective local M-CSF concentration, perturbation of the receptor-binding surface of M-CSF, and imposition of an unfavorable global orientation of the M-CSF dimer. Each prong may reduce M-CSF:FMS signaling to a limited extent but in combination may alter M-CSF:FMS signaling dramatically. The downregulating mechanism of BARF1 underlines a viral modulation strategy, and provides a basis for understanding EBV pathogenesis.

  11. Mechanism of Mitochondrial Transcription Factor A Attenuation of CpG-Induced Antibody Production

    PubMed Central

    Saifee, Jessica F.; Torres, Raul M.; Janoff, Edward N.

    2016-01-01

    Mitochondrial transcription factor A (TFAM) had previously been shown to act as a damage associated molecular pattern with the ability to enhance CpG-A phosphorothioate oligodeoxynucleotide (ODN)-mediated stimulation of IFNα production from human plasmacytoid dendritic cells. Examination of the mechanism by which TFAM might influence CpG ODN mediated innate immune responses revealed that TFAM binds directly, tightly and selectively to the structurally related CpG-A, -B, and -C ODN. TFAM also modulated the ability of the CpG-B or -C to stimulate the production of antibodies from human B cells. TFAM showed a dose-dependent modulation of CpG-B, and -C -induced antibody production from human B cells in vitro, with enhancement of high dose and inhibition of low doses of CpG stimulation. This effect was linked to the ability of TFAM to directly inhibit the binding of CpG ODNs to B cells, in a manner consistent with the relative binding affinities of TFAM for the ODNs. These data suggest that TFAM alters the free concentration of the CpG available to stimulate B cells by sequestering this ODN in a TFAM-CpG complex. Thus, TFAM has the potential to decrease the pathogenic consequences of exposure to natural CpG-like hypomethylated DNA in vivo, as well as such as that found in traumatic injury, infection, autoimmune disease and during pregnancy. PMID:27280778

  12. Knockdown of stromal interaction molecule 1 attenuates hepatocyte growth factor-induced endothelial progenitor cell proliferation.

    PubMed

    Shi, Yankun; Song, Mingbao; Guo, Ruiwei; Wang, Hong; Gao, Pan; Shi, Weibin; Huang, Lan

    2010-03-01

    Increased Ca(2+) entry through store-operated Ca(2+) channels (SOCCs) plays an essential role in the regulation of hepatocyte growth factor (HGF)-induced cell proliferation. Stromal interaction molecule 1 (STIM1) is thought to transmit endoplasmic reticulum (ER) Ca(2+) store depletion signals to the plasma membrane (PM), causing the opening of SOCCs in the PM. However, the relationship between HGF and STIM1 in endothelial progenitor cell (EPC) proliferation remains uncharacterized. The objective of this study was to evaluate the potential involvement of STIM1 in HGF-induced EPC proliferation. For this purpose, we used cultured rat bone marrow-derived EPCs and found that HGF-induced EPC proliferation at low concentrations. Store-operated Ca(2+) entry (SOCE) was elevated in HGF-treated EPCs, and the SOCC inhibitors 2-aminoethoxydiphenyl borate (2-APB) and BTP-2 inhibited the HGF-induced proliferation response. Moreover, STIM1 mRNA and protein expression levels were increased in response to HGF stimulation and knockdown of STMI1 decreased SOCE and prevented HGF-induced EPC proliferation. In conclusion, our data suggest that HGF-induced EPC proliferation is mediated partly via activation of STIM1. PMID:20404049

  13. Mesenchymal to Epithelial Transition Induced by Reprogramming Factors Attenuates the Malignancy of Cancer Cells

    PubMed Central

    Takaishi, Mikiro; Tarutani, Masahito; Takeda, Junji; Sano, Shigetoshi

    2016-01-01

    Epithelial to mesenchymal transition (EMT) is a biological process of metastatic cancer. However, an effective anticancer therapy that directly targets the EMT program has not yet been discovered. Recent studies have indicated that mesenchymal to epithelial transition (MET), the reverse phenomenon of EMT, is observed in fibroblasts during the generation of induced pluripotent stem cells. In the present study, we investigated the effects of reprogramming factors (RFs) on squamous cell carcinoma (SCC) cells. RFs-introduced cancer cells (RICs) demonstrated the enhanced epithelial characteristics in morphology with altered expression of mRNA and microRNAs. The motility and invasive activities of RICs in vitro were significantly reduced. Furthermore, xenografts of RICs exhibited no lymph node metastasis, whereas metastasis was detected in parental SCC-inoculated mice. Thus, we concluded that RICs regained epithelial properties through MET and showed reduced cancer malignancy in vitro and in vivo. Therefore, the understanding of the MET process in cancer cells by introduction of RFs may lead to the designing of a novel anticancer strategy. PMID:27258152

  14. Pf Filamentous Phage Requires UvrD for Replication in Pseudomonas aeruginosa

    PubMed Central

    Martínez, Eriel

    2016-01-01

    ABSTRACT Pf is a lysogenic filamentous phage that promotes biofilm development in Pseudomonas aeruginosa. Pf replicates by a rolling circle replication system which depends on a phage-encoded initiator protein and host factors usually involved in chromosome replication. Rep, an accessory replicative DNA helicase, is crucial for replication of filamentous phages in Escherichia coli. In contrast, here we show that, instead of depending on Rep, Pf replication depends on UvrD, an accessory helicase implicated in DNA repair. In this study, we also identified the initiator protein of Pf and found that it shares similarities with that of Vibrio phages CTXφ and VGJφ, which also depend on UvrD for replication. A structural comparative analysis of the initiator proteins of most known filamentous phages described thus far suggested that UvrD, known as a nonreplicative helicase, is involved in rolling circle replication of filamentous phages in diverse bacteria genera. This report consolidates knowledge on the new role of UvrD in filamentous phage replication, a function previously thought to be exclusive of Rep helicase. IMPORTANCE Biofilm development is a key component of the ability of Pseudomonas aeruginosa to evade host immune defenses and resist multiple drugs. Induction of the filamentous phage Pf, which usually is lysogenized in clinical and environmental isolates of P. aeruginosa, plays an important role in biofilm assembly, maturation, and dispersal. Despite the clinical relevance of Pf, the molecular biology of this phage is largely unknown. In this study, we found that rolling circle replication of Pf depends on UvrD, a DNA helicase normally involved in DNA repair. We also identified the initiator protein of Pf and found that it shares structural similarity with that of Vibrio cholerae phages CTXφ and VGJφ, which also use UvrD for replication. Our results reveal that, in addition to DNA repair, UvrD plays an essential role in rolling circle replication of

  15. Pf Filamentous Phage Requires UvrD for Replication in Pseudomonas aeruginosa.

    PubMed

    Martínez, Eriel; Campos-Gómez, Javier

    2016-01-01

    Pf is a lysogenic filamentous phage that promotes biofilm development in Pseudomonas aeruginosa. Pf replicates by a rolling circle replication system which depends on a phage-encoded initiator protein and host factors usually involved in chromosome replication. Rep, an accessory replicative DNA helicase, is crucial for replication of filamentous phages in Escherichia coli. In contrast, here we show that, instead of depending on Rep, Pf replication depends on UvrD, an accessory helicase implicated in DNA repair. In this study, we also identified the initiator protein of Pf and found that it shares similarities with that of Vibrio phages CTXφ and VGJφ, which also depend on UvrD for replication. A structural comparative analysis of the initiator proteins of most known filamentous phages described thus far suggested that UvrD, known as a nonreplicative helicase, is involved in rolling circle replication of filamentous phages in diverse bacteria genera. This report consolidates knowledge on the new role of UvrD in filamentous phage replication, a function previously thought to be exclusive of Rep helicase. IMPORTANCE Biofilm development is a key component of the ability of Pseudomonas aeruginosa to evade host immune defenses and resist multiple drugs. Induction of the filamentous phage Pf, which usually is lysogenized in clinical and environmental isolates of P. aeruginosa, plays an important role in biofilm assembly, maturation, and dispersal. Despite the clinical relevance of Pf, the molecular biology of this phage is largely unknown. In this study, we found that rolling circle replication of Pf depends on UvrD, a DNA helicase normally involved in DNA repair. We also identified the initiator protein of Pf and found that it shares structural similarity with that of Vibrio cholerae phages CTXφ and VGJφ, which also use UvrD for replication. Our results reveal that, in addition to DNA repair, UvrD plays an essential role in rolling circle replication of filamentous

  16. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation.

    PubMed

    Müller, Christine; Birmes, Franziska S; Rückert, Christian; Kalinowski, Jörn; Fetzner, Susanne

    2015-11-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s(-1), respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases. PMID:26319870

  17. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation

    PubMed Central

    Müller, Christine; Birmes, Franziska S.; Rückert, Christian; Kalinowski, Jörn

    2015-01-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s−1, respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases. PMID:26319870

  18. Nanocarrier-mediated inhibition of macrophage migration inhibitory factor attenuates secondary injury after spinal cord injury.

    PubMed

    Saxena, Tarun; Loomis, Kristin H; Pai, S Balakrishna; Karumbaiah, Lohitash; Gaupp, Eric; Patil, Ketki; Patkar, Radhika; Bellamkonda, Ravi V

    2015-02-24

    Spinal cord injury (SCI) can lead to permanent motor and sensory deficits. Following the initial traumatic insult, secondary injury mechanisms characterized by persistent heightened inflammation are initiated and lead to continued and pervasive cell death and tissue damage. Anti-inflammatory drugs such as methylprednisolone (MP) used clinically have ambiguous benefits with debilitating side effects. Typically, these drugs are administered systemically at high doses, resulting in toxicity and paradoxically increased inflammation. Furthermore, these drugs have a small time window postinjury (few hours) during which they need to be infused to be effective. As an alternative to MP, we investigated the effect of a small molecule inhibitor (Chicago sky blue, CSB) of macrophage migration inhibitory factor (MIF) for treating SCI. The pleiotropic cytokine MIF is known to contribute to upregulation of several pro-inflammatory cytokines in various disease and injury states. In vitro, CSB administration alleviated endotoxin-mediated inflammation in primary microglia and macrophages. Nanocarriers such as liposomes can potentially alleviate systemic side effects of high-dose therapy by enabling site-specific drug delivery to the spinal cord. However, the therapeutic window of 100 nm scale nanoparticle localization to the spinal cord after contusion injury is not fully known. Thus, we first investigated the ability of nanocarriers of different sizes to localize to the injured spinal cord up to 2 weeks postinjury. Results from the study showed that nanocarriers as large as 200 nm in diameter could extravasate into the injured spinal cord up to 96 h postinjury. We then formulated nanocarriers (liposomes) encapsulating CSB and administered them intravenously 48 h postinjury, within the previously determined 96 h therapeutic window. In vivo, in this clinically relevant contusion injury model in rats, CSB administration led to preservation of vascular and white matter integrity

  19. Three New Escherichia coli Phages from the Human Gut Show Promising Potential for Phage Therapy

    PubMed Central

    Dalmasso, Marion; Strain, Ronan; Neve, Horst; Franz, Charles M. A. P.; Cousin, Fabien J.; Ross, R. Paul; Hill, Colin

    2016-01-01

    With the emergence of multi-drug resistant bacteria the use of bacteriophages (phages) is gaining renewed interest as promising anti-microbial agents. The aim of this study was to isolate and characterize phages from human fecal samples. Three new coliphages, ɸAPCEc01, ɸAPCEc02 and ɸAPCEc03, were isolated. Their phenotypic and genomic characteristics, and lytic activity against biofilm, and in combination with ciprofloxacin, were investigated. All three phages reduced the growth of E. coli strain DPC6051 at multiplicity of infection (MOI) between 10−3 and 105. A cocktail of all three phages completely inhibited the growth of E. coli. The phage cocktail also reduced biofilm formation and prevented the emergence of phage-resistant mutants which occurred with single phage. When combined with ciprofloxacin, phage alone or in cocktail inhibited the growth of E. coli and prevented the emergence of resistant mutants. These three new phages are promising biocontrol agents for E. coli infections. PMID:27280590

  20. Three New Escherichia coli Phages from the Human Gut Show Promising Potential for Phage Therapy.

    PubMed

    Dalmasso, Marion; Strain, Ronan; Neve, Horst; Franz, Charles M A P; Cousin, Fabien J; Ross, R Paul; Hill, Colin

    2016-01-01

    With the emergence of multi-drug resistant bacteria the use of bacteriophages (phages) is gaining renewed interest as promising anti-microbial agents. The aim of this study was to isolate and characterize phages from human fecal samples. Three new coliphages, ɸAPCEc01, ɸAPCEc02 and ɸAPCEc03, were isolated. Their phenotypic and genomic characteristics, and lytic activity against biofilm, and in combination with ciprofloxacin, were investigated. All three phages reduced the growth of E. coli strain DPC6051 at multiplicity of infection (MOI) between 10-3 and 105. A cocktail of all three phages completely inhibited the growth of E. coli. The phage cocktail also reduced biofilm formation and prevented the emergence of phage-resistant mutants which occurred with single phage. When combined with ciprofloxacin, phage alone or in cocktail inhibited the growth of E. coli and prevented the emergence of resistant mutants. These three new phages are promising biocontrol agents for E. coli infections. PMID:27280590

  1. Tumor necrosis factor alpha transcription in macrophages is attenuated by an autocrine factor that preferentially induces NF-kappaB p50.

    PubMed

    Baer, M; Dillner, A; Schwartz, R C; Sedon, C; Nedospasov, S; Johnson, P F

    1998-10-01

    Macrophages are a major source of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), which are expressed during conditions of inflammation, infection, or injury. We identified an activity secreted by a macrophage tumor cell line that negatively regulates bacterial lipopolysaccharide (LPS)-induced expression of TNF-alpha. This activity, termed TNF-alpha-inhibiting factor (TIF), suppressed the induction of TNF-alpha expression in macrophages, whereas induction of three other proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-6, and monocyte chemoattractant protein 1) was accelerated or enhanced. A similar or identical inhibitory activity was secreted by IC-21 macrophages following LPS stimulation. Inhibition of TNF-alpha expression by macrophage conditioned medium was associated with selective induction of the NF-kappaB p50 subunit. Hyperinduction of p50 occurred with delayed kinetics in LPS-stimulated macrophages but not in fibroblasts. Overexpression of p50 blocked LPS-induced transcription from a TNF-alpha promoter reporter construct, showing that this transcription factor is an inhibitor of the TNF-alpha gene. Repression of the TNF-alpha promoter by TIF required a distal region that includes three NF-kappaB binding sites with preferential affinity for p50 homodimers. Thus, the selective repression of the TNF-alpha promoter by TIF may be explained by the specific binding of inhibitory p50 homodimers. We propose that TIF serves as a negative autocrine signal to attenuate TNF-alpha expression in activated macrophages. TIF is distinct from the known TNF-alpha-inhibiting factors IL-4, IL-10, and transforming growth factor beta and may represent a novel cytokine. PMID:9742085

  2. Tumor Necrosis Factor Alpha Transcription in Macrophages Is Attenuated by an Autocrine Factor That Preferentially Induces NF-κB p50

    PubMed Central

    Baer, Mark; Dillner, Allan; Schwartz, Richard C.; Sedon, Constance; Nedospasov, Sergei; Johnson, Peter F.

    1998-01-01

    Macrophages are a major source of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), which are expressed during conditions of inflammation, infection, or injury. We identified an activity secreted by a macrophage tumor cell line that negatively regulates bacterial lipopolysaccharide (LPS)-induced expression of TNF-α. This activity, termed TNF-α-inhibiting factor (TIF), suppressed the induction of TNF-α expression in macrophages, whereas induction of three other proinflammatory cytokines (interleukin-1β [IL-1β], IL-6, and monocyte chemoattractant protein 1) was accelerated or enhanced. A similar or identical inhibitory activity was secreted by IC-21 macrophages following LPS stimulation. Inhibition of TNF-α expression by macrophage conditioned medium was associated with selective induction of the NF-κB p50 subunit. Hyperinduction of p50 occurred with delayed kinetics in LPS-stimulated macrophages but not in fibroblasts. Overexpression of p50 blocked LPS-induced transcription from a TNF-α promoter reporter construct, showing that this transcription factor is an inhibitor of the TNF-α gene. Repression of the TNF-α promoter by TIF required a distal region that includes three NF-κB binding sites with preferential affinity for p50 homodimers. Thus, the selective repression of the TNF-α promoter by TIF may be explained by the specific binding of inhibitory p50 homodimers. We propose that TIF serves as a negative autocrine signal to attenuate TNF-α expression in activated macrophages. TIF is distinct from the known TNF-α-inhibiting factors IL-4, IL-10, and transforming growth factor β and may represent a novel cytokine. PMID:9742085

  3. Chrysin attenuates allergic airway inflammation by modulating the transcription factors T-bet and GATA-3 in mice.

    PubMed

    Du, Qiang; Gu, Xiaoyan; Cai, Jiankang; Huang, Mao; Su, Mei

    2012-07-01

    Chrysin, a flavonoid obtained from various natural sources, has been reported to possess anti-inflammatory, antitumor, antioxidant and anti-allergic activities. However, its anti-inflammatory and immunoregulatory activities in asthma animal models are poorly understood. In the present study, we examined the effects of chrysin on airway inflammation and the possible mechanisms through which it acts in a murine model of allergic asthma. BALB/c mice sensitized and challenged to ovalbumin (OVA) were administered intragastrically with chrysin at a dose of 50 mg/kg daily. Chrysin significantly suppressed OVA-induced airway hyperresponsiveness (AHR) to acetylcholine chloride (Ach). Chrysin administration significantly inhibited the total inflammatory cell and eosinophil counts in bronchoalveolar lavage fluid (BALF) and total immunoglobulin E (IgE) levels in serum. Histological examination of lung tissue demonstrated that chrysin significantly attenuated allergen-induced lung eosinophilic inflammation and mucus-producing goblet cells in the airway. In addition, chrysin triggered a switch of the immune response to allergens towards a T-helper type 1 (Th1) profile by modulating the transcription factors T-bet and GATA-3 in allergic mice. These data suggest that chrysin exhibits anti-inflammatory and immunoregulatory properties and provides new insights into the immunopharmacological role of chrysin in terms of its effects in a murine model of asthma. PMID:22552848

  4. Yi Qi Qing Re Gao Attenuates Podocyte Injury and Inhibits Vascular Endothelial Growth Factor Overexpression in Puromycin Aminonucleoside Rat Model

    PubMed Central

    Zhan, Yongli; Yang, Liping; Wen, Yumin; Liu, Huijie; Zhang, Haojun; Zhu, Bin; Han, Wenbing; Gu, Yanting; Sun, Xueyan; Dong, Xi; Zhao, Tingting; Ma, Huixia; Li, Ping

    2014-01-01

    Proteinuria is the hallmark of chronic kidney disease. Podocyte damage underlies the formation of proteinuria, and vascular endothelial growth factor (VEGF) functions as an autocrine/paracrine regulator. Yi Qi Qing Re Gao (YQQRG) has been used to treat proteinuria for more than two decades. The objective of this study was to investigate the protective effect and possible mechanisms of YQQRG on puromycin aminonucleoside (PAN) rat model. Eighty male Sprague-Dawley rats were randomized into sham group, PAN group, PAN + YQQRG group, and PAN + fosinopril group. Treatments were started 7 days before induction of nephrosis (a single intravenous injection of 40 mg/kg PAN) until day 15. 24 h urinary samples were collected on days 5, 9, and 14. The animals were sacrificed on days 3, 10, and 15, respectively. Blood samples and renal tissues were obtained for detection of biochemical and molecular biological parameters. YQQRG significantly reduced proteinuria, elevated serum albumin, and alleviated renal pathological lesions. YQQRG inhibited VEGF-A, nephrin, podocin, and CD2AP mRNA expression and elevated nephrin, podocin, and CD2AP protein levels starting on day 3. In conclusion, YQQRG attenuates podocyte injury in the rat PAN model through downregulation of VEGF-A and restoration of nephrin, podocin, and CD2AP protein expression. PMID:24963322

  5. Inhibition of Type I Insulin-Like Growth Factor Receptor Signaling Attenuates the Development of Breast Cancer Brain Metastasis

    PubMed Central

    Saldana, Sandra M.; Lee, Heng-Huan; Lowery, Frank J.; Khotskaya, Yekaterina B.; Xia, Weiya; Zhang, Chenyu; Chang, Shih-Shin; Chou, Chao-Kai; Steeg, Patricia S.; Yu, Dihua; Hung, Mien-Chie

    2013-01-01

    Brain metastasis is a common cause of mortality in cancer patients, yet potential therapeutic targets remain largely unknown. The type I insulin-like growth factor receptor (IGF-IR) is known to play a role in the progression of breast cancer and is currently being investigated in the clinical setting for various types of cancer. The present study demonstrates that IGF-IR is constitutively autophosphorylated in brain-seeking breast cancer sublines. Knockdown of IGF-IR results in a decrease of phospho-AKT and phospho-p70s6k, as well as decreased migration and invasion of MDA-MB-231Br brain-seeking cells. In addition, transient ablation of IGFBP3, which is overexpressed in brain-seeking cells, blocks IGF-IR activation. Using an in vivo experimental brain metastasis model, we show that IGF-IR knockdown brain-seeking cells have reduced potential to establish brain metastases. Finally, we demonstrate that the malignancy of brain-seeking cells is attenuated by pharmacological inhibition with picropodophyllin, an IGF-IR-specific tyrosine kinase inhibitor. Together, our data suggest that the IGF-IR is an important mediator of brain metastasis and its ablation delays the onset of brain metastases in our model system. PMID:24039934

  6. Lack of Platelet-Activating Factor Receptor Attenuates Experimental Food Allergy but Not Its Metabolic Alterations regarding Adipokine Levels

    PubMed Central

    Batista, Nathália Vieira; Fonseca, Roberta Cristelli; Perez, Denise; Pereira, Rafaela Vaz Sousa; de Lima Alves, Juliana; Pinho, Vanessa; Faria, Ana Maria Caetano; Cara, Denise Carmona

    2016-01-01

    Platelet-activating factor (PAF) is known to be an important mediator of anaphylaxis. However, there is a lack of information in the literature about the role of PAF in food allergy. The aim of this work was to elucidate the participation of PAF during food allergy development and the consequent adipose tissue inflammation along with its alterations. Our data demonstrated that, both before oral challenge and after 7 days receiving ovalbumin (OVA) diet, OVA-sensitized mice lacking the PAF receptor (PAFR) showed a decreased level of anti-OVA IgE associated with attenuated allergic markers in comparison to wild type (WT) mice. Moreover, there was less body weight and adipose tissue loss in PAFR-deficient mice. However, some features of inflamed adipose tissue presented by sensitized PAFR-deficient and WT mice after oral challenge were similar, such as a higher rate of rolling leukocytes in this tissue and lower circulating levels of adipokines (resistin and adiponectin) in comparison to nonsensitized mice. Therefore, PAF signaling through PAFR is important for the allergic response to OVA but not for the adipokine alterations caused by this inflammatory process. Our work clarifies some effects of PAF during food allergy along with its role on the metabolic consequences of this inflammatory process. PMID:27314042

  7. Anatomy of a Lactococcal Phage Tail†

    PubMed Central

    Mc Grath, Stephen; Neve, Horst; Seegers, Jos F. M. L.; Eijlander, Robyn; Vegge, Christina S.; Brøndsted, Lone; Heller, Knut J.; Fitzgerald, Gerald F.; Vogensen, Finn K.; van Sinderen, Douwe

    2006-01-01

    Bacteriophages of the Siphoviridae family utilize a long noncontractile tail to recognize, adsorb to, and inject DNA into their bacterial host. The tail anatomy of the archetypal Siphoviridae λ has been well studied, in contrast to phages infecting gram-positive bacteria. This report outlines a detailed anatomical description of a typical member of the Siphoviridae infecting a gram-positive bacterium. The tail superstructure of the lactococcal phage Tuc2009 was investigated using N-terminal protein sequencing, Western blotting, and immunogold transmission electron microscopy, allowing a tangible path to be followed from gene sequence through encoded protein to specific architectural structures on the Tuc2009 virion. This phage displays a striking parity with λ with respect to tail structure, which reenforced a model proposed for Tuc2009 tail architecture. Furthermore, comparisons with λ and other lactococcal phages allowed the specification of a number of genetic submodules likely to encode specific tail structures. PMID:16707689

  8. Tales from a thousand and one phages

    PubMed Central

    Rodriguez-Valera, Francisco; Mizuno, Carolina Megumi; Ghai, Rohit

    2014-01-01

    The sequencing of marine metagenomic fosmids led to the discovery of several new complete phage genomes. Among the 21 major sequence groups, 10 totally novel groups of marine phages could be identified. Some of these represent the first phages infecting large marine prokaryotic phyla, such as the Verrucomicrobia and the recently described Ca. Actinomarinales. Coming from a single deep photic zone sample the diversity of phages found is astonishing, and the comparison with a metavirome from the same location indicates that only 2% of the real diversity was recovered. In addition to this large macro-diversity, rich micro-diversity was also found, affecting host-recognition modules, mirroring the variation of cell surface components in their host marine microbes. PMID:24616837

  9. Comparative functional genomic analysis of Pasteurellaceae adhesins using phage display.

    PubMed

    Mullen, Lisa M; Nair, Sean P; Ward, John M; Rycroft, Andrew N; Williams, Rachel J; Henderson, Brian

    2007-05-16

    The Pasteurellaceae contain a number of important animal pathogens. Although related, the various members of this family cause a diversity of pathology in a wide variety of organ systems. Adhesion is an important virulence factor in bacterial infections. Surprisingly little is known about the adhesins of the Pasteurellaceae. To attempt to identify the genes coding for adhesins to some key components of the hosts extracellular matrix molecules, phage display libraries of fragmented genomic DNA from Haemophilus influenzae, Actinobacillus pleuropneumoniae, Pasteurella multocida and Aggregatibacter actinomycetemcomitans, were prepared in the phage display vector pG8SAET. The libraries were screened against human or porcine fibronectin, serum albumin or a commercial extracellular matrix containing type IV collagen, laminin and heparin sulphate. Four genes encoding putative adhesins were identified. These genes code for: (i) a 34 kDa human serum albumin binding protein from Haemophilus influenzae; (ii) a 12.8 kDa fibronectin-binding protein from Pasteurella multocida; (iii) a 13.7 kDa fibronectin-binding protein from A. actinomycetemcomitans; (iv) a 9.5 kDa serum albumin-binding protein from A. pleuropneumoniae. None of these genes have previously been proposed to code for adhesins. The applications of phage display with whole bacterial genomes to identify genes encoding novel adhesins in this family of bacteria are discussed. PMID:17258409

  10. Probing ADAMTS13 Substrate Specificity using Phage Display

    PubMed Central

    Desch, Karl C.; Kretz, Colin; Yee, Andrew; Gildersleeve, Robert; Metzger, Kristin; Agrawal, Nidhi; Cheng, Jane; Ginsburg, David

    2015-01-01

    Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73. PMID:25849793

  11. Supersize me: Cronobacter sakazakii phage GAP32

    SciTech Connect

    Abbasifar, Reza; Griffiths, Mansel W.; Sabour, Parviz M.; Ackermann, Hans-Wolfgang; Vandersteegen, Katrien; Lavigne, Rob; Noben, Jean-Paul; Alanis Villa, Argentina; Abbasifar, Arash; Nash, John H.E.; Kropinski, Andrew M.

    2014-07-15

    Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB{sub C}saM{sub G}AP32 (GAP32), which possesses the second largest sequenced phage genome (358,663 bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB{sub K}leM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily. - Highlights: • Cronobacter sakazakii phage vB{sub C}saM{sub G}AP32 has a genome of 358,663 bp. • It encodes 545 proteins which is more than Mycoplasma genitalium G37. • It is a member of the Myoviridae. • It is peripherally related to coliphage PBECO4 and Klebsiella phage vB{sub K}leM-RaK2. • GAP32 encodes a chromosome condensation protein.

  12. Dynamic and static light scattering analysis of DNA ejection from the phage λ

    NASA Astrophysics Data System (ADS)

    Löf, David; Schillén, Karin; Jönsson, Bengt; Evilevitch, Alex

    2007-07-01

    With the aid of time-resolved dynamic light scattering (DLS) and static light scattering (SLS), we have analyzed the ejection kinetics from the bacterial virus bacteriophage (or phage) λ , triggered in vitro by its receptor. We have used DLS to investigate the kinetics in such a system. Furthermore, we have shown that both SLS and DLS can be interchangeably used to study the process of phage DNA release. DLS is superior to SLS in that it also allows the change in the light scattering arising from each of the components in the system to be monitored under conditions such that the relaxation times are separable. With help of these two methods we present a model explaining the reason for the observed decrease in the scattering intensity accompanying DNA ejection from phage. We emphasize that ejection from phage capsid occurs through a very long tail (which is nearly three times longer than the capsid diameter), which significantly separates ejected DNA from the scattering volume of the capsid. The scattering intensity recorded during the DNA ejection process is the result of a change in the form factor of the phage particle, i.e., the change in the interference effects between the phage capsid and the DNA confined in the phage particle. When the DNA molecule is completely ejected it remains in the proximity of the phage for some time, thus contributing to the scattering signal as it diffuses away from the phage capsid, into the scattering volume and returns to its unperturbed chain conformation in bulk solution. The free DNA chain does not contribute to the scattered intensity, when measured at a large angle, due to the DNA form factor and the low concentration. Although the final diffusion-controlled step can lead to overestimation of the real ejection time, we can still use both scattering methods to estimate the initial DNA ejection rates, which are mainly dependent on the pressure-driven DNA ejection from the phage, allowing studies of the effects of various

  13. Ovarian gonadotrophin surge-attenuating factor (GnSAF): where are we after 20 years of research?

    PubMed

    Fowler, Paul A; Sorsa-Leslie, Tarja; Harris, William; Mason, Helen D

    2003-12-01

    When gonadotrophin-stimulated IVF methods were being developed in the 1970s and 1980s, understanding of the physiology of FSH improved. In addition to its classic actions of stimulating aromatase activity and oestradiol secretion by ovarian granulosa cells, FSH was found to stimulate the ovarian production of an uncharacterized hormone known by its specific effect of reducing pituitary responsiveness to GnRH. This hormone has been called gonadotrophin surge-attenuating factor (GnSAF), gonadotropin surge-inhibiting factor (GnSIF), various abbreviations (GnSAF/IF, GnSIF/AF) and also attenuin. Although first described in the 1980s, GnSAF has still not been convincingly characterized and no published candidate amino acid sequences conclusively relate to GnSAF bioactivity. On the basis of superovulation studies and in vitro experimentation into the roles of steroids in regulating LH, GnRH and GnRH self-priming, the concept that GnSAF has a role in the regulation of LH secretion, the timing of the LH surge and the prevention of premature luteinization developed. For at least a decade, understanding of the specific GnSAF effects of reducing pituitary sensitivity to GnRH, especially GnRH self-priming and antagonizing the stimulatory effects of oestradiol on GnRH-induced LH secretion, supported this concept. However, improved knowledge of the changes in GnSAF bioactivity in follicular fluid and serum in women requires revision of this concept. The present authors propose that the main role of GnSAF is probably the negative regulation of pulsatile LH secretion, mainly during the first half of the follicular phase, indicating a critical role in the regulation of folliculogenesis and oestradiol secretion. PMID:14748688

  14. Epidermal growth factor attenuates tubular necrosis following mercuric chloride damage by regeneration of indigenous, not bone marrow-derived cells

    PubMed Central

    Yen, Tzung-Hai; Alison, Malcolm R; Goodlad, Robert A; Otto, William R; Jeffery, Rosemary; Cook, H Terence; Wright, Nicholas A; Poulsom, Richard

    2015-01-01

    To assess effects of epidermal growth factor (EGF) and pegylated granulocyte colony-stimulating factor (P-GCSF; pegfilgrastim) administration on the cellular origin of renal tubular epithelium regenerating after acute kidney injury initiated by mercuric chloride (HgCl2). Female mice were irradiated and male whole bone marrow (BM) was transplanted into them. Six weeks later recipient mice were assigned to one of eight groups: control, P-GCSF+, EGF+, P-GCSF+EGF+, HgCl2, HgCl2+P-GCSF+, HgCl2+EGF+ and HgCl2+P-GCSF+EGF+. Following HgCl2, injection tubular injury scores increased and serum urea nitrogen levels reached uraemia after 3 days, but EGF-treated groups were resistant to this acute kidney injury. A four-in-one analytical technique for identification of cellular origin, tubular phenotype, basement membrane and S-phase status revealed that BM contributed 1% of proximal tubular epithelium in undamaged kidneys and 3% after HgCl2 damage, with no effects of exogenous EGF or P-GCSF. Only 0.5% proximal tubular cells were seen in S-phase in the undamaged group kidneys; this increased to 7–8% after HgCl2 damage and to 15% after addition of EGF. Most of the regenerating tubular epithelium originated from the indigenous pool. BM contributed up to 6.6% of the proximal tubular cells in S-phase after HgCl2 damage, but only to 3.3% after additional EGF. EGF administration attenuated tubular necrosis following HgCl2 damage, and the major cause of this protective effect was division of indigenous cells, whereas BM-derived cells were less responsive. P-GCSF did not influence damage or regeneration. PMID:25389045

  15. Vitamin K3 attenuates lipopolysaccharide-induced acute lung injury through inhibition of nuclear factor-κB activation

    PubMed Central

    Tanaka, S; Nishiumi, S; Nishida, M; Mizushina, Y; Kobayashi, K; Masuda, A; Fujita, T; Morita, Y; Mizuno, S; Kutsumi, H; Azuma, T; Yoshida, M

    2010-01-01

    Vitamin K is a family of fat-soluble compounds including phylloquinone (vitamin K1), menaquinone (vitamin K2) and menadione (vitamin K3). Recently, it was reported that vitamin K, especially vitamins K1 and K2, exerts a variety of biological effects, and these compounds are expected to be candidates for therapeutic agents against various diseases. In this study, we investigated the anti-inflammatory effects of vitamin K3 in in vitro cultured cell experiments and in vivo animal experiments. In human embryonic kidney (HEK)293 cells, vitamin K3 inhibited the tumour necrosis factor (TNF)-α-evoked translocation of nuclear factor (NF)-κB into the nucleus, although vitamins K1 and K2 did not. Vitamin K3 also suppressed the lipopolysaccharide (LPS)-induced nuclear translocation of NF-κB and production of TNF-α in mouse macrophage RAW264·7 cells. Moreover, the addition of vitamin K3 before and after LPS administration attenuated the severity of lung injury in an animal model of acute lung injury/acute respiratory distress syndrome (ARDS), which occurs in the setting of acute severe illness complicated by systemic inflammation. In the ARDS model, vitamin K3 also suppressed the LPS-induced increase in the serum TNF-α level and inhibited the LPS-evoked nuclear translocation of NF-κB in lung tissue. Despite marked efforts, little therapeutic progress has been made, and the mortality rate of ARDS remains high. Vitamin K3 may be an effective therapeutic strategy against acute lung injury including ARDS. PMID:20030669

  16. Diversity and censoring of landscape phage libraries

    PubMed Central

    Kuzmicheva, G.A.; Jayanna, P.K.; Sorokulova, I.B.; Petrenko, V.A.

    2009-01-01

    Libraries of random peptides displayed on the surface of filamentous phages are a valuable source for biospecific ligands. However, their successful use can be hindered by a disproportionate representation of different phage clones and fluctuation of their composition that arises during phage reproduction, which have potential to affect efficiency of selection of clones with an optimal binding. Therefore, there is a need to develop phage display libraries with extended and varied repertoires of displayed peptides. In this work, we compared the complexity, evolution and representation of two phage display libraries displaying foreign octamers and nonamers in 4000 copies as the N-terminal part of the major coat protein pVIII of phage fd–tet (landscape libraries). They were obtained by replacement of amino acids 2–4 and 2–5 of pVIII with random octa- and nonamers, respectively. Statistical analysis of the libraries revealed their dramatic censoring and evolution during amplification. Further, a survey of both libraries for clones that bind common selectors revealed the presence of different non-overlapping families of target-specific clones in each library justifying the concept that different landscape libraries cover different areas of a sequence space. PMID:18988692

  17. Diversity and censoring of landscape phage libraries.

    PubMed

    Kuzmicheva, G A; Jayanna, P K; Sorokulova, I B; Petrenko, V A

    2009-01-01

    Libraries of random peptides displayed on the surface of filamentous phages are a valuable source for biospecific ligands. However, their successful use can be hindered by a disproportionate representation of different phage clones and fluctuation of their composition that arises during phage reproduction, which have potential to affect efficiency of selection of clones with an optimal binding. Therefore, there is a need to develop phage display libraries with extended and varied repertoires of displayed peptides. In this work, we compared the complexity, evolution and representation of two phage display libraries displaying foreign octamers and nonamers in 4000 copies as the N-terminal part of the major coat protein pVIII of phage fd-tet (landscape libraries). They were obtained by replacement of amino acids 2-4 and 2-5 of pVIII with random octa- and nonamers, respectively. Statistical analysis of the libraries revealed their dramatic censoring and evolution during amplification. Further, a survey of both libraries for clones that bind common selectors revealed the presence of different non-overlapping families of target-specific clones in each library justifying the concept that different landscape libraries cover different areas of a sequence space. PMID:18988692

  18. Down regulation of virulence factors of Pseudomonas aeruginosa by salicylic acid attenuates its virulence on Arabidopsis thaliana and Caenorhabditis elegans.

    PubMed

    Prithiviraj, B; Bais, H P; Weir, T; Suresh, B; Najarro, E H; Dayakar, B V; Schweizer, H P; Vivanco, J M

    2005-09-01

    Salicylic acid (SA) is a phenolic metabolite produced by plants and is known to play an important role in several physiological processes, such as the induction of plant defense responses against pathogen attack. Here, using the Arabidopsis thaliana-Pseudomonas aeruginosa pathosystem, we provide evidence that SA acts directly on the pathogen, down regulating fitness and virulence factor production of the bacteria. Pseudomonas aeruginosa PA14 showed reduced attachment and biofilm formation on the roots of the Arabidopsis mutants lox2 and cpr5-2, which produce elevated amounts of SA, as well as on wild-type Arabidopsis plants primed with exogenous SA, a treatment known to enhance endogenous SA concentration. Salicylic acid at a concentration that did not inhibit PA14 growth was sufficient to significantly affect the ability of the bacteria to attach and form biofilm communities on abiotic surfaces. Furthermore, SA down regulated three known virulence factors of PA14: pyocyanin, protease, and elastase. Interestingly, P. aeruginosa produced more pyocyanin when infiltrated into leaves of the Arabidopsis transgenic line NahG, which accumulates less SA than wild-type plants. This finding suggests that endogenous SA plays a role in down regulating the synthesis and secretion of pyocyanin in vivo. To further test if SA directly affects the virulence of P. aeruginosa, we used the Caenorhabditis elegans-P. aeruginosa infection model. The addition of SA to P. aeruginosa lawns significantly diminished the bacterium's ability to kill the worms, without affecting the accumulation of bacteria inside the nematodes' guts, suggesting that SA negatively affects factors that influence the virulence of P. aeruginosa. We employed microarray technology to identify SA target genes. These analyses showed that SA treatment affected expression of 331 genes. It selectively repressed transcription of exoproteins and other virulence factors, while it had no effect on expression of housekeeping

  19. [Lytic phages and prophages of Streptococcus suis--a review].

    PubMed

    Tang, Fang; Lu, Chengping

    2015-04-01

    Streptococcus suis (S. suis) is an important zoonosis and pathogen that can carry prophages. In this review, we focus on the recent advances in our understanding of lytic phage and lysogenic phage of S. suis, including the morphology of S. suis lytic phage, the functions of lysin and terminase large subunit encoded by S. suis lytic phage, comparative genomics of S. suis prophages, lysogenic. conversion between S. suis lytic phage and prophage. Furthermore, prospective evolution of interactions between phage and host was discussed. PMID:26211312

  20. Attenuation of atherosclerotic lesions in diabetic apolipoprotein E-deficient mice using gene silencing of macrophage migration inhibitory factor

    PubMed Central

    Sun, Hui; Zhang, XianJun; Zhao, Lei; Zhen, Xi; Huang, ShanYing; Wang, ShaSha; He, Hong; Liu, ZiMo; Xu, NaNa; Yang, FaLin; Qu, ZhongHua; Ma, ZhiYong; Zhang, Cheng; Zhang, Yun; Hu, Qin

    2015-01-01

    Macrophage migration inhibitory factor (MIF) involves the pathogenesis of atherosclerosis (AS) and increased plasma MIF levels in diabetes mellitus (DM) patients are associated with AS. Here, we have been suggested that MIF could be a critical contributor for the pathological process of diabetes-associated AS by using adenovirus-mediated RNA interference. First, streptozotocin (STZ)-induced diabetic animal model was constructed in 114 apolipoprotein E-deficient mice (apoE−/− mice) fed on a regular chow diet. Then, the animals were randomly divided into three groups: Adenovirus-mediated MIF interference (Ad-MIFi), Ad-enhanced green fluorescent protein (EGFP) and normal saline (NS) group (n ≈ 33/group). Non-diabetic apoE−/− mice (n = 35) were served as controls. Ad-MIFi, Ad-EGFP and NS were, respectively, injected into the tail vein of mice from Ad-MIFi, Ad-EGFP and NS group, which were injected repeatedly 4 weeks later. Physical, biochemical, morphological and molecular parameters were measured. The results showed that diabetic apoE−/− mice had significantly aggravated atherosclerotic lesions. MIF gene interference attenuated atherosclerotic lesions and stabilized atheromatous plaque, accompanied by the decreased macrophages and lipids deposition and inflammatory cytokines production, improved glucose intolerance and plasma cholesterol level, the decreased ratio of matrix matalloproteinase-2/tissue inhibitor of metalloproteinase-1 and plaque instability index. An increased expression of MIF and its ligand CD74 was also detected in the diabetic patients with coronary artery disease. The results suggest that MIF gene interference is able to inhibit atherosclerotic lesions and increase plaque stability in diabetic apoE−/−mice. MIF inhibition could be a novel and promising approach to the treatment of DM-associated AS. PMID:25661015

  1. Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells.

    PubMed

    Dushpanova, Anar; Agostini, Silvia; Ciofini, Enrica; Cabiati, Manuela; Casieri, Valentina; Matteucci, Marco; Del Ry, Silvia; Clerico, Aldo; Berti, Sergio; Lionetti, Vincenzo

    2016-01-01

    Expression of endothelin (ET)-1 is increased in endothelial cells exposed to angiotensin II (Ang II), leading to endothelial dysfunction and cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents Ang II-induced ET-1 upregulation. Nearly 65 ± 2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with vWF-specific siRNA without affecting cell viability and growth. While showing ET-1 similar to wild type cells at rest, vWF-silenced cells did not present ET-1 upregulation during exposure to Ang II (100 nM/24 h), preserving levels of endothelial nitric oxide synthase activity similar to wild type. vWF silencing prevented AngII-induced increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity and superoxide anion (O2-) levels, known triggers of ET-1 expression. Moreover, no increase in O2- or ET-1 levels was found in silenced cells treated with AngII or NOX-agonist phorbol ester (PMA 5 nM/48 h). Finally, vWF was required for overexpression of NOX4 and NOX2 in response to AngII and PMA. In conclusion, endothelial vWF knockdown prevented Ang II-induced ET-1 upregulation through attenuation of NOX-mediated O2- production. Our findings reveal a new role of vWF in preventing of Ang II-induced endothelial dysfunction. PMID:27443965

  2. Genetic Inhibition of Fibroblast Growth Factor Receptor 1 in Knee Cartilage Attenuates the Degeneration of Articular Cartilage in Adult Mice

    PubMed Central

    Weng, Tujun; Yi, Lingxian; Huang, Junlan; Luo, Fengtao; Wen, Xuan; Du, Xiaolan; Chen, Qian; Deng, Chuxia; Chen, Di; Chen, Lin

    2013-01-01

    Objective Fibroblast growth factor (FGF) family members are involved in the regulation of articular cartilage homeostasis. The aim of this study was to investigate the function of FGF receptor 1 (FGFR-1) in the development of osteoarthritis (OA) and its underlying mechanisms. Methods FGFR-1 was deleted from the articular chondrocytes of adult mice in a cartilage-specific and tamoxifen-inducible manner. Two OA models (aging-associated spontaneous OA, and destabilization-induced OA), as well as an antigen-induced arthritis (AIA) model, were established and tested in Fgfr1-deficient and wild-type (WT) mice. Alterations in cartilage structure and the loss of proteoglycan were assessed in the knee joints of mice of either genotype, using these 3 arthritis models. Primary chondrocytes were isolated and the expression of key regulatory molecules was assessed quantitatively. In addition, the effect of an FGFR-1 inhibitor on human articular chondrocytes was examined. Results The gross morphologic features of Fgfr1-deficient mice were comparable with those of WT mice at both the postnatal and adult stages. The articular cartilage of 12-month-old Fgfr1-deficient mice displayed greater aggrecan staining compared to 12-month-old WT mice. Fgfr1 deficiency conferred resistance to the proteoglycan loss induced by AIA and attenuated the development of cartilage destruction after surgically induced destabilization of the knee joint. The chondroprotective effect of FGFR-1 inhibition was largely associated with decreased expression of matrix metalloproteinase 13 (MMP-13) and up-regulation of FGFR-3 in mouse and human articular chondrocytes. Conclusion Disruption of FGFR-1 in adult mouse articular chondrocytes inhibits the progression of cartilage degeneration. Down-regulation of MMP-13 expression and up-regulation of FGFR-3 levels may contribute to the phenotypic changes observed in Fgfr1-deficient mice. PMID:22833219

  3. Aminoguanidine and curcumin attenuated tumor necrosis factor (TNF)-α-induced oxidative stress, colitis and hepatotoxicity in mice.

    PubMed

    Mouzaoui, Souad; Rahim, Ibtissem; Djerdjouri, Bahia

    2012-01-01

    The up regulation of gut mucosal cytokines such as tumor necrosis factor (TNF)-α and oxidative stress have been related to inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This study investigated an immune-mediated model of colitis. TNF-α injected intraperitonally to mice induced a dose-dependent recruitment of neutrophils into abdominal mesentery. The leukocytes influx induced by TNF-α (10 μg kg(-1) body weight) increased by 3 fold liver and colon damage scores. TNF-α-colitis was characterized by hemorrhagic edemas and crypt abscesses massively infiltrated by inflammatory cells, namely neutrophils. Moreover, TNF-α-toxicity resulted in liver steatosis and foci of necrosis infiltrated by Kupffer cells and neutrophils in parenchyma and around the centrilobular veins. The involvement of oxidative stress was evaluated using aminoguanidine (AG) as selective inhibitor of inducible NO synthase (iNOS) and curcumin (Cur), the polyphenolic antioxidant of turmeric (Curcuma longa L.). TNF-α-toxicity led to significant increase in myeloperoxidase (MPO, an index of neutrophils infiltration), nitrites (stable nitric oxide metabolites) and malondialdehyde (MDA, a marker of lipid peroxides) levels and cell apoptosis in liver and colon. AG and Cur treatments significantly attenuated the hallmarks of oxidative stress, neutrophils influx and ROS-related cellular and histological damages, in TNF-α-treated mice. Taken together, our results provide insights into the role of phagocytes-derived oxidants in TNF-α-colitis in mice. Cur and AG, by inhibiting neutrophils priming and iNOsynthase could be effective against oxidative bowel damages induced in IBD by imbalanced gut immune response. PMID:22036766

  4. Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells

    PubMed Central

    Dushpanova, Anar; Agostini, Silvia; Ciofini, Enrica; Cabiati, Manuela; Casieri, Valentina; Matteucci, Marco; Del Ry, Silvia; Clerico, Aldo; Berti, Sergio; Lionetti, Vincenzo

    2016-01-01

    Expression of endothelin (ET)-1 is increased in endothelial cells exposed to angiotensin II (Ang II), leading to endothelial dysfunction and cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents Ang II-induced ET-1 upregulation. Nearly 65 ± 2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with vWF-specific siRNA without affecting cell viability and growth. While showing ET-1 similar to wild type cells at rest, vWF-silenced cells did not present ET-1 upregulation during exposure to Ang II (100 nM/24 h), preserving levels of endothelial nitric oxide synthase activity similar to wild type. vWF silencing prevented AngII-induced increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity and superoxide anion (O2−) levels, known triggers of ET-1 expression. Moreover, no increase in O2− or ET-1 levels was found in silenced cells treated with AngII or NOX-agonist phorbol ester (PMA 5 nM/48 h). Finally, vWF was required for overexpression of NOX4 and NOX2 in response to AngII and PMA. In conclusion, endothelial vWF knockdown prevented Ang II-induced ET-1 upregulation through attenuation of NOX-mediated O2− production. Our findings reveal a new role of vWF in preventing of Ang II-induced endothelial dysfunction. PMID:27443965

  5. Genome analysis of the staphylococcal temperate phage DW2 and functional studies on the endolysin and tail hydrolase

    PubMed Central

    Keary, Ruth; McAuliffe, Olivia; Ross, R Paul; Hill, Colin; O’Mahony, Jim; Coffey, Aidan

    2014-01-01

    This study describes the genome of temperate Siphoviridae phage DW2, which is routinely propagated on Staphylococcus aureus DPC5246. The 41941 bp genome revealed an open reading frame (ORF1) which has a high level of homology with members of the resolvase subfamily of site-specific serine recombinase, involved in chromosomal integration and excision. In contrast, the majority of staphylococcal phages reported to date encode tyrosine recombinases. Two putative genes encoded by phage DW2 (ORF15 and ORF24) were highly homologous to the NWMN0273 and NWMN0280 genes encoding virulence factors carried on the genome of ϕNM4, a prophage in the genome of S. aureus Newman. Phage DW2 also encodes proteins highly homologous to two well-characterized Staphylococcus aureus pathogenicity island derepressors encoded by the staphylococcal helper phage 80α indicating that it may similarly act as a helper phage for mobility of pathogenicity islands in S. aureus. This study also focused on the enzybiotic potential of phage DW2. The structure of the putative endolysin and tail hydrolase were investigated and used as the basis for a cloning strategy to create recombinant peptidoglycan hydrolyzing proteins. After overexpression in E. coli, four of these proteins (LysDW2, THDW2, CHAPE1-153, and CHAPE1-163) were demonstrated to have hydrolytic activity against peptidoglycan of S. aureus and thus represent novel candidates for exploitation as enzybiotics. PMID:25105056

  6. Burkholderia cepacia complex Phage-Antibiotic Synergy (PAS): antibiotics stimulate lytic phage activity.

    PubMed

    Kamal, Fatima; Dennis, Jonathan J

    2015-02-01

    The Burkholderia cepacia complex (Bcc) is a group of at least 18 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. Bcc organisms possess high levels of innate antimicrobial resistance, and alternative therapeutic strategies are urgently needed. One proposed alternative treatment is phage therapy, the therapeutic application of bacterial viruses (or bacteriophages). Recently, some phages have been observed to form larger plaques in the presence of sublethal concentrations of certain antibiotics; this effect has been termed phage-antibiotic synergy (PAS). Those reports suggest that some antibiotics stimulate increased production of phages under certain conditions. The aim of this study is to examine PAS in phages that infect Burkholderia cenocepacia strains C6433 and K56-2. Bcc phages KS12 and KS14 were tested for PAS, using 6 antibiotics representing 4 different drug classes. Of the antibiotics tested, the most pronounced effects were observed for meropenem, ciprofloxacin, and tetracycline. When grown with subinhibitory concentrations of these three antibiotics, cells developed a chain-like arrangement, an elongated morphology, and a clustered arrangement, respectively. When treated with progressively higher antibiotic concentrations, both the sizes of plaques and phage titers increased, up to a maximum. B. cenocepacia K56-2-infected Galleria mellonella larvae treated with phage KS12 and low-dose meropenem demonstrated increased survival over controls treated with KS12 or antibiotic alone. These results suggest that antibiotics can be combined with phages to stimulate increased phage production and/or activity and thus improve the efficacy of bacterial killing. PMID:25452284

  7. Estimation of Path Length Reduction Factor by Using One Year Rain Attenuation Statistics over a Line of Sight Link Operating at 28.75 GHz in Amritsar (INDIA)

    NASA Astrophysics Data System (ADS)

    Sharma, Parshotam; Hudiara, Inderjit Singh; Singh, Maninder Lal

    2011-02-01

    The effect of environmental factors in general and rain droplets in particular, on microwave propagation is a very well known fact now. If the rain droplets are present in an inhomogeneous way across the path length of the microwave communication system then, a new concept of path length reduction factor is introduced which accounts for the inhomogeneous nature of the rain droplets along the path length of the microwave signal. The present paper presents results of path length reduction factor using data on attenuation levels obtained on a LOS link operating at 28.75 GHz in Amritsar region and its comparison with Crane's and ITU-R's model.

  8. Commensal E. coli Stx2 lysogens produce high levels of phages after spontaneous prophage induction

    PubMed Central

    Iversen, Hildegunn; L' Abée-Lund, Trine M.; Aspholm, Marina; Arnesen, Lotte P. S.; Lindbäck, Toril

    2015-01-01

    Enterohemorrhagic E. coli (EHEC) is a food-borne pathogen that causes disease ranging from uncomplicated diarrhea to life-threatening hemolytic uremic syndrome (HUS) and nervous system complications. Shiga toxin 2 (Stx2) is the major virulence factor of EHEC and is critical for development of HUS. The genes encoding Stx2 are carried by lambdoid bacteriophages and the toxin production is tightly linked to the production of phages during lytic cycle. It has previously been suggested that commensal E. coli could amplify the production of Stx2-phages and contribute to the severity of disease. In this study we examined the susceptibility of commensal E. coli strains to the Stx2-converting phage ϕ734, isolated from a highly virulent EHEC O103:H25 (NIPH-11060424). Among 38 commensal E. coli strains from healthy children below 5 years, 15 were lysogenized by the ϕ734 phage, whereas lytic infection was not observed. Three of the commensal E. coli ϕ734 lysogens were tested for stability, and appeared stable and retained the phage for at least 10 cultural passages. When induced to enter lytic cycle by H2O2 treatment, 8 out of 13 commensal lysogens produced more ϕ734 phages than NIPH-11060424. Strikingly, five of them even spontaneously (non-induced) produced higher levels of phage than the H2O2 induced NIPH-11060424. An especially high frequency of HUS (60%) was seen among children infected by NIPH-11060424 during the outbreak in 2006. Based on our findings, a high Stx2 production by commensal E. coli lysogens cannot be ruled out as a contributor to the high frequency of HUS during this outbreak. PMID:25692100

  9. Gnotobiotic mouse model of phage-bacterial host dynamics in the human gut.

    PubMed

    Reyes, Alejandro; Wu, Meng; McNulty, Nathan P; Rohwer, Forest L; Gordon, Jeffrey I

    2013-12-10

    Bacterial viruses (phages) are the most abundant biological group on Earth and are more genetically diverse than their bacterial prey/hosts. To characterize their role as agents shaping gut microbial community structure, adult germ-free mice were colonized with a consortium of 15 sequenced human bacterial symbionts, 13 of which harbored one or more predicted prophages. One member, Bacteroides cellulosilyticus WH2, was represented by a library of isogenic transposon mutants that covered 90% of its genes. Once assembled, the community was subjected to a staged phage attack with a pool of live or heat-killed virus-like particles (VLPs) purified from the fecal microbiota of five healthy humans. Shotgun sequencing of DNA from the input pooled VLP preparation plus shotgun sequencing of gut microbiota samples and purified fecal VLPs from the gnotobiotic mice revealed a reproducible nonsimultaneous pattern of attack extending over a 25-d period that involved five phages, none described previously. This system allowed us to (i) correlate increases in specific phages present in the pooled VLPs with reductions in the representation of particular bacterial taxa, (ii) provide evidence that phage resistance occurred because of ecological or epigenetic factors, (iii) track the origin of each of the five phages among the five human donors plus the extent of their genome variation between and within recipient mice, and (iv) establish the dramatic in vivo fitness advantage that a locus within a B. cellulosilyticus prophage confers upon its host. Together, these results provide a defined community-wide view of phage-bacterial host dynamics in the gut. PMID:24259713

  10. Commensal E. coli Stx2 lysogens produce high levels of phages after spontaneous prophage induction.

    PubMed

    Iversen, Hildegunn; L' Abée-Lund, Trine M; Aspholm, Marina; Arnesen, Lotte P S; Lindbäck, Toril

    2015-01-01

    Enterohemorrhagic E. coli (EHEC) is a food-borne pathogen that causes disease ranging from uncomplicated diarrhea to life-threatening hemolytic uremic syndrome (HUS) and nervous system complications. Shiga toxin 2 (Stx2) is the major virulence factor of EHEC and is critical for development of HUS. The genes encoding Stx2 are carried by lambdoid bacteriophages and the toxin production is tightly linked to the production of phages during lytic cycle. It has previously been suggested that commensal E. coli could amplify the production of Stx2-phages and contribute to the severity of disease. In this study we examined the susceptibility of commensal E. coli strains to the Stx2-converting phage ϕ734, isolated from a highly virulent EHEC O103:H25 (NIPH-11060424). Among 38 commensal E. coli strains from healthy children below 5 years, 15 were lysogenized by the ϕ734 phage, whereas lytic infection was not observed. Three of the commensal E. coli ϕ734 lysogens were tested for stability, and appeared stable and retained the phage for at least 10 cultural passages. When induced to enter lytic cycle by H2O2 treatment, 8 out of 13 commensal lysogens produced more ϕ734 phages than NIPH-11060424. Strikingly, five of them even spontaneously (non-induced) produced higher levels of phage than the H2O2 induced NIPH-11060424. An especially high frequency of HUS (60%) was seen among children infected by NIPH-11060424 during the outbreak in 2006. Based on our findings, a high Stx2 production by commensal E. coli lysogens cannot be ruled out as a contributor to the high frequency of HUS during this outbreak. PMID:25692100

  11. Characterization of Two Virulent Phages of Lactobacillus plantarum

    PubMed Central

    Briggiler Marcó, Mariángeles; Garneau, Josiane E.; Tremblay, Denise; Quiberoni, Andrea

    2012-01-01

    We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria. PMID:23042172

  12. The tail-associated depolymerase of Erwinia amylovora phage L1 mediates host cell adsorption and enzymatic capsule removal, which can enhance infection by other phage.

    PubMed

    Born, Yannick; Fieseler, Lars; Klumpp, Jochen; Eugster, Marcel R; Zurfluh, Katrin; Duffy, Brion; Loessner, Martin J

    2014-07-01

    The depolymerase enzyme (DpoL1) encoded by the T7-like phage L1 efficiently degrades amylovoran, an important virulence factor and major component of the extracellular polysaccharide (EPS) of its host, the plant pathogen Erwinia amylovora. Mass spectrometry analysis of hydrolysed EPS revealed that DpoL1 cleaves the galactose-containing backbone of amylovoran. The enzyme is most active at pH 6 and 50°C, and features a modular architecture. Removal of 180 N-terminal amino acids was shown not to affect enzyme activity. The C-terminus harbours the hydrolase activity, while the N-terminal domain links the enzyme to the phage particle. Electron microscopy demonstrated that DpoL1-specific antibodies cross-link phage particles at their tails, either lateral or frontal, and immunogold staining confirmed that DpoL1 is located at the tail spikes. Exposure of high-level EPS-producing Er. amylovora strain CFBP1430 to recombinant DpoL1 dramatically increased sensitivity to the Dpo-negative phage Y2, which was not the case for EPS-negative mutants or low-level EPS-producing Er. amylovora. Our findings indicate that enhanced phage susceptibility is based on enzymatic removal of the EPS capsule, normally a physical barrier to Y2 infection, and that use of DpoL1 together with the broad host range, virulent phage Y2 represents an attractive combination for biocontrol of fire blight. PMID:23944160

  13. Sulforaphane attenuates hepatic fibrosis via NF-E2-related factor 2-mediated inhibition of transforming growth factor-β/Smad signaling.

    PubMed

    Oh, Chang Joo; Kim, Joon-Young; Min, Ae-Kyung; Park, Keun-Gyu; Harris, Robert A; Kim, Han-Jong; Lee, In-Kyu

    2012-02-01

    Sulforaphane (SFN) is a dietary isothiocyanate that exerts chemopreventive effects via NF-E2-related factor 2 (Nrf2)-mediated induction of antioxidant/phase II enzymes, such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). This work was undertaken to evaluate the effects of SFN on hepatic fibrosis and profibrotic transforming growth factor (TGF)-β/Smad signaling, which are closely associated with oxidative stress. SFN suppressed TGF-β-enhanced expression of α-smooth muscle actin (α-SMA), a marker of hepatic stellate cell (HSC) activation, and profibrogenic genes such as type I collagen, fibronectin, tissue inhibitor of matrix metalloproteinase (TIMP)-1, and plasminogen activator inhibitor (PAI)-1 in hTERT, an immortalized human HSC line. SFN inhibited TGF-β-stimulated activity of a PAI-1 promoter construct and (CAGA)(9) MLP-Luc, an artificial Smad3/4-specific reporter, in addition to reducing phosphorylation and nuclear translocation of Smad3. Nrf2 overexpression was sufficient to inhibit the TGF-β/Smad signaling and PAI-1 expression. Conversely, knockdown of Nrf2, but not inhibition of HO-1 or NQO1 activity, significantly abolished the inhibitory effect of SFN on (CAGA)(9) MLP-Luc activity. However, inhibition of NQO1 activity reversed repression of TGF-β-stimulated expression of type I collagen by SFN, suggesting the involvement of antioxidant activity of SFN in the suppression of Smad-independent fibrogenic gene expression. Finally, SFN treatment attenuated the development and progression of early stage hepatic fibrosis induced by bile duct ligation in mice, accompanied by reduced expression of type I collagen and α-SMA. Collectively, these results show that SFN elicits an antifibrotic effect on hepatic fibrosis through Nrf2-mediated inhibition of the TGF-β/Smad signaling and subsequent suppression of HSC activation and fibrogenic gene expression. PMID:22155056

  14. Repeated intravenous administrations of teneurin-C terminal associated peptide (TCAP)-1 attenuates reinstatement of cocaine seeking by corticotropin-releasing factor (CRF) in rats.

    PubMed

    Erb, Suzanne; McPhee, Matthew; Brown, Zenya J; Kupferschmidt, David A; Song, Lifang; Lovejoy, David A

    2014-08-01

    The teneurin c-terminal associated peptides (TCAP) have been implicated in the regulation of the stress response, possibly via a corticotropin-releasing factor (CRF)-related mechanism. We have previously shown that repeated intracerebroventricular (ICV) injections of TCAP-1 attenuate the reinstatement of cocaine seeking by CRF in rats. Here, we determined whether intravenous (IV) administrations of TCAP-1 would likewise attenuate CRF-induced reinstatement, and whether this effect would vary depending on the rat's history of cocaine self administration. Rats were trained to self-administer cocaine for 10 days, during once daily sessions that were either 3h ("short access"; ShA) or 6h ("long access"; LgA). Rats were then given five daily injections of TCAP-1 (0, 300, or 3,000 pmol, IV) in their home cage. Subsequently, they were returned to the self-administration chambers where extinction of cocaine seeking and testing for CRF-induced reinstatement of cocaine seeking was carried out. Repeated IV administrations of TCAP-1 were efficacious in attenuating CRF-induced reinstatement of cocaine seeking, but at different doses in ShA and LgA rats. Taken together, the findings extend previous work showing a consistent effect of repeated ICV TCAP-1 on CRF-induced reinstatement of cocaine seeking, and point to a potential therapeutic benefit of TCAP-1 in attenuating cocaine seeking behaviors. PMID:24768621

  15. Targeting Enterococcus faecalis biofilms with phage therapy.

    PubMed

    Khalifa, Leron; Brosh, Yair; Gelman, Daniel; Coppenhagen-Glazer, Shunit; Beyth, Shaul; Poradosu-Cohen, Ronit; Que, Yok-Ai; Beyth, Nurit; Hazan, Ronen

    2015-04-01

    Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment. PMID:25662974

  16. Evidence for metaviromic islands in marine phages

    PubMed Central

    Mizuno, Carolina Megumi; Ghai, Rohit; Rodriguez-Valera, Francisco

    2014-01-01

    Metagenomic islands (MGIs) have been defined as genomic regions in prokaryotic genomes that under-recruit from metagenomes where most of the same genome recruits at close to 100% identity over most of its length. The presence of MGIs in prokaryotes has been associated to the diversity of concurrent lineages that vary at this level to disperse the predatory pressure of phages that, reciprocally, maintain high clonal diversity in the population and improve ecosystem performance. This was proposed as a Constant-Diversity (C-D) model. Here we have investigated the regions of phage genomes under-recruiting in a metavirome constructed with a sample from the same habitat where they were retrieved. Some of the genes found to under-recruit are involved in host recognition as would be expected from the C-D model. Furthermore, the recruitment of intragenic regions known to be involved in molecular recognition also had a significant under-recruitment compared to the rest of the gene. However, other genes apparently disconnected from the recognition process under-recruited often, specifically the terminases involved in packaging of the phage genome in the capsid and a few others. In addition, some highly related phage genomes (at nucleotide sequence level) had no metaviromic islands (MVIs). We speculate that the latter might be generalist phages with broad infection range that do not require clone specific lineages. PMID:24550898

  17. Targeting Enterococcus faecalis Biofilms with Phage Therapy

    PubMed Central

    Khalifa, Leron; Brosh, Yair; Gelman, Daniel; Coppenhagen-Glazer, Shunit; Beyth, Shaul; Poradosu-Cohen, Ronit; Que, Yok-Ai; Beyth, Nurit

    2015-01-01

    Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment. PMID:25662974

  18. Antibody phage display technology and its applications.

    PubMed

    Hoogenboom, H R; de Bruïne, A P; Hufton, S E; Hoet, R M; Arends, J W; Roovers, R C

    1998-06-01

    In recent years, the use of display vectors and in vitro selection technologies has transformed the way in which we generate ligands, such as antibodies and peptides, for a given target. Using this technology, we are now able to design repertoires of ligands from scratch and use the power of phage selection to select those ligands having the desired (biological) properties. With phage display, tailor-made antibodies may be synthesized and selected to acquire the desired affinity of binding and specificity for in vitro and in vivo diagnosis, or for immunotherapy of human disease. This review addresses recent progress in the construction of, and selection from phage antibody libraries, together with novel approaches for screening phage antibodies. As the quality of large naïve and synthetic antibody repertoires improves and libraries becomes more generally available, new and exciting applications are pioneered such as the identification of novel antigens using differential selection and the generation of receptor a(nta)gonists. A combination of the design and generation of millions to billions of different ligands, together with phage display for the isolation of binding ligands and with functional assays for identifying (and possibly selecting) bio-active ligands, will open even more challenging applications of this inspiring technology, and provide a powerful tool for drug and target discovery well into the next decade. PMID:9661810

  19. Current taxonomy of phages infecting lactic acid bacteria

    PubMed Central

    Mahony, Jennifer; van Sinderen, Douwe

    2013-01-01

    Phages infecting lactic acid bacteria have been the focus of significant research attention over the past three decades. Through the isolation and characterization of hundreds of phage isolates, it has been possible to classify phages of the dairy starter and adjunct bacteria Lactococus lactis, Streptococcus thermophilus, Leuconostoc spp., and Lactobacillus spp. Among these, phages of L. lactis have been most thoroughly scrutinized and serve as an excellent model system to address issues that arise when attempting taxonomic classification of phages infecting other LAB species. Here, we present an overview of the current taxonomy of phages infecting LAB genera of industrial significance, the methods employed in these taxonomic efforts and how these may be employed for the taxonomy of phages of currently underrepresented and emerging phage species. PMID:24478767

  20. Phages of Listeria offer novel tools for diagnostics and biocontrol

    PubMed Central

    Hagens, Steven; Loessner, Martin J.

    2014-01-01

    Historically, bacteriophages infecting their hosts have perhaps been best known and even notorious for being a nuisance in dairy-fermentation processes. However, with the rapid progress in molecular microbiology and microbial ecology, a new dawn has risen for phages. This review will provide an overview on possible uses and applications of Listeria phages, including phage-typing, reporter phage for bacterial diagnostics, and use of phage as biocontrol agents for food safety. The use of phage-encoded enzymes such as endolysins for the detection and as antimicrobial agent will also be addressed. Desirable properties of candidate phages for biocontrol will be discussed. While emphasizing the enormous future potential for applications, we will also consider some of the intrinsic limitations dictated by both phage and bacterial ecology. PMID:24782847

  1. Aerosol Phage Therapy Efficacy in Burkholderia cepacia Complex Respiratory Infections

    PubMed Central

    Semler, Diana D.; Goudie, Amanda D.; Finlay, Warren H.

    2014-01-01

    Phage therapy has been suggested as a potential treatment for highly antibiotic-resistant bacteria, such as the species of the Burkholderia cepacia complex (BCC). To address this hypothesis, experimental B. cenocepacia respiratory infections were established in mice using a nebulizer and a nose-only inhalation device. Following infection, the mice were treated with one of five B. cenocepacia-specific phages delivered as either an aerosol or intraperitoneal injection. The bacterial and phage titers within the lungs were assayed 2 days after treatment, and mice that received the aerosolized phage therapy demonstrated significant decreases in bacterial loads. Differences in phage activity were observed in vivo. Mice that received phage treatment by intraperitoneal injection did not demonstrate significantly reduced bacterial loads, although phage particles were isolated from their lung tissue. Based on these data, aerosol phage therapy appears to be an effective method for treating highly antibiotic-resistant bacterial respiratory infections, including those caused by BCC bacteria. PMID:24798268

  2. The Significance of Mutualistic Phages for Bacterial Ecology and Evolution.

    PubMed

    Obeng, Nancy; Pratama, Akbar Adjie; Elsas, Jan Dirk van

    2016-06-01

    Bacteria and phages have traditionally been viewed as 'antagonists'. However, temperate phages can transfer genes, which can broaden their bacterial hosts' metabolic repertoire, confer or enhance virulence, or eliminate competing organisms, and so enhance bacterial fitness. Recent evidence shows that phages can also promote biofilm formation leading to population-level benefits for their bacterial hosts. Here, we provide a perspective on the ecological and evolutionary consequences for the bacteria interacting with phages, when phage and host interests are aligned. Furthermore, we examine the question whether bacterial hosts can lower immune barriers to phage infection, thereby facilitating infection by beneficial phages. Taking recent evidence together, we suggest that in many cases temperate phages are to be considered as being mutualistic as well as parasitic, at the same time. PMID:26826796

  3. The gastrointestinal phage communities of the cultivated freshwater fishes.

    PubMed

    He, Yang; Yang, Hongjiang

    2015-03-01

    The phage communities in the gut of 62 cultivated freshwater fish were investigated by culture-based methods. Using three selective media, 445 pathogenic bacilli strains were isolated and used as indicators for subsequent phage isolations. Totally, 63 phages were detected and the respective host strains were identified with the comparative sequence analysis of 16S rRNA gene, including Aeromonas (29), Vibrio (1), Citrobacter (16), Serratia (4), Enterobacter (2), Proteus (3), Buttiauxella (2), Plesiomonas (2), Kluyvera (1), Morgenella (2) and Providencia (1). The diversity of Aeromonas phages was assessed by discrimination of their host strains with random amplified polymorphic DNA method. Furthermore, the isolated Aeromonas phages were characterized by host range and growth inhibition assay. The results demonstrated that there were abundant and diverse phage populations in the gut environment of the cultivated freshwater fishes. The phages could contribute to the microbiota balance in the gut ecosystem of fishes and provide reliable phage sources for future applications. PMID:25743067

  4. Aerosol phage therapy efficacy in Burkholderia cepacia complex respiratory infections.

    PubMed

    Semler, Diana D; Goudie, Amanda D; Finlay, Warren H; Dennis, Jonathan J

    2014-07-01

    Phage therapy has been suggested as a potential treatment for highly antibiotic-resistant bacteria, such as the species of the Burkholderia cepacia complex (BCC). To address this hypothesis, experimental B. cenocepacia respiratory infections were established in mice using a nebulizer and a nose-only inhalation device. Following infection, the mice were treated with one of five B. cenocepacia-specific phages delivered as either an aerosol or intraperitoneal injection. The bacterial and phage titers within the lungs were assayed 2 days after treatment, and mice that received the aerosolized phage therapy demonstrated significant decreases in bacterial loads. Differences in phage activity were observed in vivo. Mice that received phage treatment by intraperitoneal injection did not demonstrate significantly reduced bacterial loads, although phage particles were isolated from their lung tissue. Based on these data, aerosol phage therapy appears to be an effective method for treating highly antibiotic-resistant bacterial respiratory infections, including those caused by BCC bacteria. PMID:24798268

  5. Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei.

    PubMed

    Mercanti, Diego J; Rousseau, Geneviève M; Capra, María L; Quiberoni, Andrea; Tremblay, Denise M; Labrie, Simon J; Moineau, Sylvain

    2016-01-01

    Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: CL1, CL2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (CL1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages CL1, CL2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages CL1, CL2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts. PMID:26475105

  6. Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei

    PubMed Central

    Rousseau, Geneviève M.; Capra, María L.; Quiberoni, Andrea; Tremblay, Denise M.; Labrie, Simon J.

    2015-01-01

    Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: CL1, CL2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (CL1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages CL1, CL2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages CL1, CL2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts. PMID:26475105

  7. Phage display and Shiga toxin neutralizers.

    PubMed

    Bernedo-Navarro, Robert Alvin; Yano, Tomomasa

    2016-04-01

    The current work presents an overview of the use of phage display technology for the identification and characterization of potential neutralizing agents for Shiga toxins. The last major Shiga toxin-associated disease outbreak, which took place in Germany in 2011, showed the international community that Shiga toxins remain a serious threat to public health. This is also demonstrated by the lack of specific therapies against Shiga toxin-induced Hemolytic Uremic Syndrome (HUS). Since its inception, phage display technology has played a key role in the development of antigen-specific (poly)-peptides or antibody fragments with specific biological properties. Herein, we review the current literature regarding the application of phage display to identify novel neutralizing agents against Shiga toxins. We also briefly highlight reported discoveries of peptides and heavy chain antibodies (VHH fragments or nanobodies) that can neutralize the cellular damage caused by these potent toxins. PMID:26898657

  8. Phage therapy in the food industry.

    PubMed

    Endersen, Lorraine; O'Mahony, Jim; Hill, Colin; Ross, R Paul; McAuliffe, Olivia; Coffey, Aidan

    2014-01-01

    Despite advances in modern technologies, the food industry is continuously challenged with the threat of microbial contamination. The overuse of antibiotics has further escalated this problem, resulting in the increasing emergence of antibiotic-resistant foodborne pathogens. Efforts to develop new methods for controlling microbial contamination in food and the food processing environment are extremely important. Accordingly, bacteriophages (phages) and their derivatives have emerged as novel, viable, and safe options for the prevention, treatment, and/or eradication of these contaminants in a range of foods and food processing environments. Whole phages, modified phages, and their derivatives are discussed in terms of current uses and future potential as antimicrobials in the traditional farm-to-fork context, encompassing areas such as primary production, postharvest processing, biosanitation, and biodetection. The review also presents some safety concerns to ensure safe and effective exploitation of bacteriophages in the future. PMID:24422588

  9. Direct measurement via phage titre of the dissociation constants in solution of fusion phage-substrate complexes.

    PubMed Central

    Dyson, M R; Germaschewski, V; Murray, K

    1995-01-01

    Studies of interactions between filamentous fusion phage particles and protein or nucleic acid molecules have gained increasing importance with recent successes of screening techniques based upon random phage display libraries (biopanning). Since a number of different phage are usually obtained by biopanning, it is useful to compare quantitatively the binding affinities of individual phage for the substrate used for selection. A procedure is described for determination of relative dissociation constants (KdRel) between filamentous phage carrying peptide fusions to the coat protein gpIII and substrates in solution. This novel method is based on the measurement of phage titres. Phage selected from a random fusion phage library for binding to a monoclonal antibody or a viral structural protein exhibited KdRel values in the nanomolar and micromolar ranges for their respective substrates, thus validating the method over a wide range of binding affinities. PMID:7784206

  10. Complete Genome Sequence of Pseudomonas aeruginosa Phage AAT-1

    PubMed Central

    Andrade-Domínguez, Andrés

    2016-01-01

    Aspects of the interaction between phages and animals are of interest and importance for medical applications. Here, we report the genome sequence of the lytic Pseudomonas phage AAT-1, isolated from mammalian serum. AAT-1 is a double-stranded DNA phage, with a genome of 57,599 bp, containing 76 predicted open reading frames. PMID:27563032

  11. HostPhinder: A Phage Host Prediction Tool

    PubMed Central

    Villarroel, Julia; Kleinheinz, Kortine Annina; Jurtz, Vanessa Isabell; Zschach, Henrike; Lund, Ole; Nielsen, Morten; Larsen, Mette Voldby

    2016-01-01

    The current dramatic increase of antibiotic resistant bacteria has revitalised the interest in bacteriophages as alternative antibacterial treatment. Meanwhile, the development of bioinformatics methods for analysing genomic data places high-throughput approaches for phage characterization within reach. Here, we present HostPhinder, a tool aimed at predicting the bacterial host of phages by examining the phage genome sequence. Using a reference database of 2196 phages with known hosts, HostPhinder predicts the host species of a query phage as the host of the most genomically similar reference phages. As a measure of genomic similarity the number of co-occurring k-mers (DNA sequences of length k) is used. Using an independent evaluation set, HostPhinder was able to correctly predict host genus and species for 81% and 74% of the phages respectively, giving predictions for more phages than BLAST and significantly outperforming BLAST on phages for which both had predictions. HostPhinder predictions on phage draft genomes from the INTESTI phage cocktail corresponded well with the advertised targets of the cocktail. Our study indicates that for most phages genomic similarity correlates well with related bacterial hosts. HostPhinder is available as an interactive web service [1] and as a stand alone download from the Docker registry [2]. PMID:27153081

  12. Genome Sequences of Gordonia Phages Bowser and Schwabeltier

    PubMed Central

    Montgomery, Matthew T.; Arnold, Zachary M.; Basina, Aleksandra; Iyer, Ankitha M.; Stoner, Ty H.; Kasturiarachi, Naomi S.; Pressimone, Catherine A.; Schiebel, Johnathon G.; Furbee, Emily C.; Grubb, Sarah R.; Warner, Marcie H.; Garlena, Rebecca A.; Russell, Daniel A.; Jacobs-Sera, Deborah; Hatfull, Graham F.

    2016-01-01

    Gordonia phages Bowser and Schwabeltier are newly isolated phages infecting Gordonia terrae 3612. Bowser and Schwabeltier have similar siphoviral morphologies and their genomes are related to each other, but not to other phages. Their lysis cassettes are atypically situated among virion tail genes, and Bowser encodes two tyrosine integrases. PMID:27516498

  13. HostPhinder: A Phage Host Prediction Tool.

    PubMed

    Villarroel, Julia; Kleinheinz, Kortine Annina; Jurtz, Vanessa Isabell; Zschach, Henrike; Lund, Ole; Nielsen, Morten; Larsen, Mette Voldby

    2016-01-01

    The current dramatic increase of antibiotic resistant bacteria has revitalised the interest in bacteriophages as alternative antibacterial treatment. Meanwhile, the development of bioinformatics methods for analysing genomic data places high-throughput approaches for phage characterization within reach. Here, we present HostPhinder, a tool aimed at predicting the bacterial host of phages by examining the phage genome sequence. Using a reference database of 2196 phages with known hosts, HostPhinder predicts the host species of a query phage as the host of the most genomically similar reference phages. As a measure of genomic similarity the number of co-occurring k-mers (DNA sequences of length k) is used. Using an independent evaluation set, HostPhinder was able to correctly predict host genus and species for 81% and 74% of the phages respectively, giving predictions for more phages than BLAST and significantly outperforming BLAST on phages for which both had predictions. HostPhinder predictions on phage draft genomes from the INTESTI phage cocktail corresponded well with the advertised targets of the cocktail. Our study indicates that for most phages genomic similarity correlates well with related bacterial hosts. HostPhinder is available as an interactive web service [1] and as a stand alone download from the Docker registry [2]. PMID:27153081

  14. Complete Genome Sequence of Pseudomonas aeruginosa Phage AAT-1.

    PubMed

    Andrade-Domínguez, Andrés; Kolter, Roberto

    2016-01-01

    Aspects of the interaction between phages and animals are of interest and importance for medical applications. Here, we report the genome sequence of the lytic Pseudomonas phage AAT-1, isolated from mammalian serum. AAT-1 is a double-stranded DNA phage, with a genome of 57,599 bp, containing 76 predicted open reading frames. PMID:27563032

  15. Measurement of the x-ray mass attenuation coefficient and the imaginary part of the form factor of silicon using synchrotron radiation

    NASA Astrophysics Data System (ADS)

    Tran, C. Q.; Chantler, C. T.; Barnea, Z.; Paterson, D.; Cookson, D. J.

    2003-04-01

    We used the x-ray extended-range technique to measure the x-ray mass attenuation coefficients of silicon with an accuracy between 0.27% and 0.5% in the 5 keV-20 keV energy range. Subtraction of the x-ray scattering contribution enabled us to derive the corresponding x-ray photoelectric absorption coefficients and determine the absolute value of the imaginary part of the atomic form factor of silicon. Discrepancies between the experimental values of the mass attenuation coefficients and theoretically calculated values are discussed. New approaches to the theoretical calculation will be required to match the precision and accuracy of the experimental results.

  16. Growth Hormone-Releaser Diet Attenuates Cognitive Dysfunction in Klotho Mutant Mice via Insulin-Like Growth Factor-1 Receptor Activation in a Genetic Aging Model

    PubMed Central

    Park, Seok Joo; Chung, Yoon Hee; Lee, Jeong Hyun; Dang, Duy-Khanh; Nam, Yunsung; Jeong, Ji Hoon; Kim, Yong Sun; Nabeshima, Toshitaka

    2014-01-01

    Background It has been recognized that a defect in klotho gene expression accelerates the degeneration of multiple age-sensitive traits. Accumulating evidence indicates that aging is associated with declines in cognitive function and the activity of growth hormone (GH)/insulin-like growth factor-1 (IGF-1). Methods In this study, we examined whether a GH-releaser diet could be effective in protecting against cognitive impairment in klotho mutant mice. Results The GH-releaser diet significantly induced the expression of IGF-1 and IGF-1 receptors in the hippocampus of klotho mutant mice. Klotho mutant mice showed significant memory impairments as compared with wild-type mice. In addition, the klotho mutation significantly decreased the expression of cell survival/antiapoptotic factors, including phospho-Akt (p-Akt)/phospho-glycogen synthase kinase3β (p-GSK3β), phospho-extracellular signal-related kinase (p-ERK), and Bcl-2, but significantly increased those of cell death/proapoptotic factors, such as phospho-c-jun N-terminal kinase (p-JNK), Bax, and cleaved caspase-3 in the hippocampus. Treatment with GH-releaser diet significantly attenuated both decreases in the expression of cell survival/antiapoptotic factors and increases in the expression of cell death/proapoptotic factors in the hippocampus of klotho mutant mice. In addition, klotho mutation-induced oxidative stress was significantly attenuated by the GH-releaser diet. Consequently, a GH-releaser diet significantly improved memory function in the klotho mutant mice. GH-releaser diet-mediated actions were significantly reversed by JB-1, an IGF-1 receptor antagonist. Conclusion The results suggest that a GH-releaser diet attenuates oxidative stress, proapoptotic changes and consequent dysfunction in klotho mutant mice by promoting IGF-1 expression and IGF-1 receptor activation. PMID:25309793

  17. Diversity and geographical distribution of Flavobacterium psychrophilum isolates and their phages: patterns of susceptibility to phage infection and phage host range.

    PubMed

    Castillo, Daniel; Christiansen, Rói Hammershaimb; Espejo, Romilio; Middelboe, Mathias

    2014-05-01

    Flavobacterium psychrophilum is an important fish pathogen worldwide that causes cold water disease (CWD) or rainbow trout fry syndrome (RTFS). Phage therapy has been suggested as an alternative method for the control of this pathogen in aquaculture. However, effective use of bacteriophages in disease control requires detailed knowledge about the diversity and dynamics of host susceptibility to phage infection. For this reason, we examined the genetic diversity of 49 F. psychrophilum strains isolated in three different areas (Chile, Denmark, and USA) through direct genome restriction enzyme analysis (DGREA) and their susceptibility to 33 bacteriophages isolated in Chile and Denmark, thus covering large geographical (>12,000 km) and temporal (>60 years) scales of isolation. An additional 40 phage-resistant isolates obtained from culture experiments after exposure to specific phages were examined for changes in phage susceptibility against the 33 phages. The F. psychrophilum and phage populations isolated from Chile and Denmark clustered into geographically distinct groups with respect to DGREA profile and host range, respectively. However, cross infection between Chilean phage isolates and Danish host isolates and vice versa was observed. Development of resistance to certain bacteriophages led to susceptibility to other phages suggesting that "enhanced infection" is potentially an important cost of resistance in F. psychrophilum, possibly contributing to the observed co-existence of phage-sensitive F. psychrophilum strains and lytic phages across local and global scales. Overall, our results showed that despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be globally distributed. PMID:24557506

  18. Insulinlike Growth Factor I Plus Insulinlike Growth Factor Binding Protein 3 Attenuates the Proinflammatory Acute Phase Response in Severely Burned Children

    PubMed Central

    Jeschke, Marc G.; Barrow, Robert E.; Herndon, David N.

    2000-01-01

    Objective To determine the effect of insulinlike growth factor I (IGF-I) in combination with its principal binding protein (IGFBP-3) on the hepatic acute phase response in severely burned children. Summary Background Data The hepatic acute phase response is a cascade of events initiated to restore homeostasis after trauma. A prolonged response, however, may contribute to multiple organ failure, hypermetabolism, complications, and death. Methods Twenty-two children with a mean total body surface area (TBSA) burn of 57 ± 3% were given a continuous infusion of 1 to 4 mg/kg/day IGF-I/BP-3 for 5 days after wound excision and grafting. Eight children with a TBSA burn of 54 ± 4% were given saline as controls. Before and 5 days after excision and grafting, blood samples were taken for serum hepatic constitutive protein, acute phase protein, and proinflammatory cytokine analysis. Results Serum IGF-I levels in burned children given the IGF-I/BP-3 complex increased from 113 ± 15 to 458 ± 40 ng/mL and IGFBP-3 levels increased from 1.8 ± 0.2 to 3.1 ± 0.3 ng/mL. Levels of serum constitutive hepatic proteins (prealbumin, retinol-binding protein, and transferrin) increased with IGF-I/BP-3, whereas levels of type I acute phase proteins (C-reactive protein, α1-acid glycoprotein, and complement C-3) decreased when compared with controls. The complex had no effect on type II acute phase proteins. Tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) levels decreased with IGF-I/BP-3 compared with controls, with no effect on interleukin-6. Conclusion Severely burned children receiving IGF-I/BP-3 showed a decrease in IL-1β and TNF-α followed by a decrease in type I acute phase proteins that was associated with a concomitant increase in constitutive hepatic proteins. Attenuating the proinflammatory acute phase with IGF-1/BP-3 response may prevent multiple organ failure and improve clinical outcomes after thermal injury without any detectable adverse side effects. PMID

  19. Chopping-Wheel Optical Attenuator

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B.

    1988-01-01

    Star-shaped rotating chopping wheel provides adjustable time-averaged attenuation of narrow beam of light without changing length of optical path or spectral distribution of light. Duty cycle or attenuation factor of chopped beam controlled by adjusting radius at which beam intersects wheel. Attenuation factor independent of wavelength. Useful in systems in which chopping frequency above frequency-response limits of photodetectors receiving chopped light. Used in systems using synchronous detection with lock-in amplifiers.

  20. Phage & phosphatase: a novel phage-based probe for rapid, multi-platform detection of bacteria.

    PubMed

    Alcaine, S D; Pacitto, D; Sela, D A; Nugen, S R

    2015-11-21

    Genetic engineering of bacteriophages allows for the development of rapid, highly specific, and easily manufactured probes for the detection of bacterial pathogens. A challenge for novel probes is the ease of their adoption in real world laboratories. We have engineered the bacteriophage T7, which targets Escherichia coli, to carry the alkaline phosphatase gene, phoA. This inclusion results in phoA overexpression following phage infection of E. coli. Alkaline phosphatase is commonly used in a wide range of diagnostics, and thus a signal produced by our phage-based probe could be detected using common laboratory equipment. Our work demonstrates the successful: (i) modification of T7 phage to carry phoA; (ii) overexpression of alkaline phosphatase in E. coli; and (iii) detection of this T7-induced alkaline phosphatase activity using commercially available colorimetric and chemilumiscent methods. Furthermore, we demonstrate the application of our phage-based probe to rapidly detect low levels of bacteria and discern the antibiotic resistance of E. coli isolates. Using our bioengineered phage-based probe we were able to detect 10(3) CFU per mL of E. coli in 6 hours using a chemiluminescent substrate and 10(4) CFU per mL within 7.5 hours using a colorimetric substrate. We also show the application of this phage-based probe for antibiotic resistance testing. We were able to determine whether an E. coli isolate was resistant to ampicillin within 4.5 hours using chemiluminescent substrate and within 6 hours using a colorimetric substrate. This phage-based scheme could be readily adopted in labs without significant capital investments and can be translated to other phage-bacteria pairs for further detection. PMID:26421320

  1. Phage therapies for plants and people.

    PubMed

    Gross, Michael

    2014-06-16

    The use of bacteriophages to combat bacterial infections may help to address the current crisis of antibiotic resistance. Fundamental issues arising from the ecological dynamic of host, bacterium and phage can be investigated in trees, offering both a natural approach to treating plant disease, and a chance to avoid creating a new resistance problem. Michael Gross reports. PMID:25075391

  2. Morphological evidence for phages of Xylella fastidiosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Through transmission electron microscopy (TEM), phage particles of Xylella fastidiosa strain Temecula-1 grown in PW broth were observed in ultrathin sections of bacterial cell-containing low speed centrifugation pellets and in partially purified preparations from CsCl equilibrium centrifugation dens...

  3. Interaction Analysis through Proteomic Phage Display

    PubMed Central

    2014-01-01

    Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance. PMID:25295249

  4. Interaction analysis through proteomic phage display.

    PubMed

    Sundell, Gustav N; Ivarsson, Ylva

    2014-01-01

    Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance. PMID:25295249

  5. Genome Sequence of Gordonia Phage Emalyn

    PubMed Central

    Guido, Madeline J.; Iyengar, Pragnya; Nigra, Jonathan T.; Serbin, Matthew B.; Kasturiarachi, Naomi S.; Pressimone, Catherine A.; Schiebel, Johnathon G.; Furbee, Emily C.; Grubb, Sarah R.; Warner, Marcie H.; Montgomery, Matthew T.; Garlena, Rebecca A.; Russell, Daniel A.; Jacobs-Sera, Deborah; Hatfull, Graham F.

    2016-01-01

    Emalyn is a newly isolated bacteriophage of Gordonia terrae 3612 and has a double-stranded DNA genome 43,982 bp long with 67 predicted protein-encoding genes, 32 of which we can assign putative functions. Emalyn has a prolate capsid and has extensive nucleotide similarity with several previously sequenced phages. PMID:27516499

  6. Genome Sequence of Gordonia Phage Emalyn.

    PubMed

    Pope, Welkin H; Guido, Madeline J; Iyengar, Pragnya; Nigra, Jonathan T; Serbin, Matthew B; Kasturiarachi, Naomi S; Pressimone, Catherine A; Schiebel, Johnathon G; Furbee, Emily C; Grubb, Sarah R; Warner, Marcie H; Montgomery, Matthew T; Garlena, Rebecca A; Russell, Daniel A; Jacobs-Sera, Deborah; Hatfull, Graham F

    2016-01-01

    Emalyn is a newly isolated bacteriophage of Gordonia terrae 3612 and has a double-stranded DNA genome 43,982 bp long with 67 predicted protein-encoding genes, 32 of which we can assign putative functions. Emalyn has a prolate capsid and has extensive nucleotide similarity with several previously sequenced phages. PMID:27516499

  7. DeltaPhage--a novel helper phage for high-valence pIX phagemid display.

    PubMed

    Nilssen, Nicolay R; Frigstad, Terje; Pollmann, Sylvie; Roos, Norbert; Bogen, Bjarne; Sandlie, Inger; Løset, Geir Å

    2012-09-01

    Phage display has been instrumental in discovery of novel binding peptides and folded domains for the past two decades. We recently reported a novel pIX phagemid display system that is characterized by a strong preference for phagemid packaging combined with low display levels, two key features that support highly efficient affinity selection. However, high diversity in selected repertoires are intimately coupled to high display levels during initial selection rounds. To incorporate this additional feature into the pIX display system, we have developed a novel helper phage termed DeltaPhage that allows for high-valence display on pIX. This was obtained by inserting two amber mutations close to the pIX start codon, but after the pVII translational stop, conditionally inactivating the helper phage encoded pIX. Until now, the general notion has been that display on pIX is dependent on wild-type complementation, making high-valence display unachievable. However, we found that DeltaPhage does facilitate high-valence pIX display when used with a non-suppressor host. Here, we report a side-by-side comparison with pIII display, and we find that this novel helper phage complements existing pIX phagemid display systems to allow both low and high-valence display, making pIX display a complete and efficient alternative to existing pIII phagemid display systems. PMID:22539265

  8. Phage-bacteria interaction network in human oral microbiome.

    PubMed

    Wang, Jinfeng; Gao, Yuan; Zhao, Fangqing

    2016-07-01

    Although increasing knowledge suggests that bacteriophages play important roles in regulating microbial ecosystems, phage-bacteria interaction in human oral cavities remains less understood. Here we performed a metagenomic analysis to explore the composition and variation of oral dsDNA phage populations and potential phage-bacteria interaction. A total of 1,711 contigs assembled with more than 100 Gb shotgun sequencing data were annotated to 104 phages based on their best BLAST matches against the NR database. Bray-Curtis dissimilarities demonstrated that both phage and bacterial composition are highly diverse between periodontally healthy samples but show a trend towards homogenization in diseased gingivae samples. Significantly, according to the CRISPR arrays that record infection relationship between bacteria and phage, we found certain oral phages were able to invade other bacteria besides their putative bacterial hosts. These cross-infective phages were positively correlated with commensal bacteria while were negatively correlated with major periodontal pathogens, suggesting possible connection between these phages and microbial community structure in oral cavities. By characterizing phage-bacteria interaction as networks rather than exclusively pairwise predator-prey relationships, our study provides the first insight into the participation of cross-infective phages in forming human oral microbiota. PMID:26036920

  9. Characterization and adsorption of Lactobacillus virulent phage P1.

    PubMed

    Chen, X; Xi, Y; Zhang, H; Wang, Z; Fan, M; Liu, Y; Wu, W

    2016-09-01

    Bacteriophage infection of lactic acid bacteria is considered an important problem worldwide in the food fermentation industry, as it may produce low quality or unsafe foods, cause fermentation failure, and result in economic losses. To increase current knowledge on the properties of Lactobacillus virulent phages, we evaluated the effect of divalent cations, temperature, pH, and chloramphenicol on the adsorption ability of Lactobacillus virulent phage P1. Phage P1 was isolated from the abnormal fermentation liquid of Lactobacillus plantarum IMAU10120. The results showed that this phage belonged to the Siphoviridae family. The latent period of this phage was 45min, and the burst time was 90min. Burst size was 132.88±2.37 phage counts expressed per milliliter per infective center. This phage showed good tolerance at different temperatures, but incubation at 50°C only affected its adsorption. Adsorption rate reached a maximum value between 30 and 42°C. A high adsorption value of phage infectivity was obtained from pH 6 to 8. Moreover, calcium ions promoted and increased the adsorption capacity of phage P1, but magnesium ions had negative effects. Chloramphenicol had no effect on phage adsorption. This study increased current knowledge on the characterization and biological aspects of Lactobacillus virulent phages, and may provide some basic information that can be used to design successful antiphage strategies in the food industry. PMID:27372579

  10. Phage-bacterial interactions in the evolution of toxigenic Vibrio cholerae.

    PubMed

    Faruque, Shah M; Mekalanos, John J

    2012-11-15

    Understanding the genetic and ecological factors which support the emergence of new clones of pathogenic bacteria is vital to develop preventive measures. Vibrio cholerae the causative agent of cholera epidemics represents a paradigm for this process in that this organism evolved from environmental non-pathogenic strains by acquisition of virulence genes. The major virulence factors of V. cholerae, cholera toxin (CT) and toxin coregulated pilus (TCP) are encoded by a lysogenic bacteriophage (CTXφ) and a pathogenicity island, respectively. Additional phages which cooperate with the CTXφ in horizontal transfer of genes in V. cholerae have been characterized, and the potential exists for discovering yet new phages or genetic elements which support the transfer of genes for environmental fitness and virulence leading to the emergence of new epidemic strains. Phages have also been shown to play a crucial role in modulating seasonal cholera epidemics. Thus, the complex array of natural phenomena driving the evolution of pathogenic V. cholerae includes, among other factors, phages that either participate in horizontal gene transfer or in a bactericidal selection process favoring the emergence of new clones of V. cholerae. PMID:23076327

  11. Twelve previously unknown phage genera are ubiquitous in global oceans

    SciTech Connect

    Holmfeldt, Karin; Solonenko, Natalie; Shah, Manesh B; Corrier, Kristen L; Riemann, Lasse; Verberkmoes, Nathan C; Sullivan, Matthew B

    2013-01-01

    Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in unknowns dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four wellknown viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria. Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage host systems for experimental hypothesis testing.

  12. Twelve previously unknown phage genera are ubiquitous in global oceans.

    PubMed

    Holmfeldt, Karin; Solonenko, Natalie; Shah, Manesh; Corrier, Kristen; Riemann, Lasse; Verberkmoes, Nathan C; Sullivan, Matthew B

    2013-07-30

    Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in "unknowns" dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four well-known viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria. Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage-host systems for experimental hypothesis testing. PMID:23858439

  13. Artificial Neural Networks Trained to Detect Viral and Phage Structural Proteins

    PubMed Central

    Seguritan, Victor; Raymond, Amy; Lorimer, Don; Burgin, Alex B.; Salamon, Peter; Segall, Anca M.

    2012-01-01

    Phages play critical roles in the survival and pathogenicity of their hosts, via lysogenic conversion factors, and in nutrient redistribution, via cell lysis. Analyses of phage- and viral-encoded genes in environmental samples provide insights into the physiological impact of viruses on microbial communities and human health. However, phage ORFs are extremely diverse of which over 70% of them are dissimilar to any genes with annotated functions in GenBank. Better identification of viruses would also aid in better detection and diagnosis of disease, in vaccine development, and generally in better understanding the physiological potential of any environment. In contrast to enzymes, viral structural protein function can be much more challenging to detect from sequence data because of low sequence conservation, few known conserved catalytic sites or sequence domains, and relatively limited experimental data. We have designed a method of predicting phage structural protein sequences that uses Artificial Neural Networks (ANNs). First, we trained ANNs to classify viral structural proteins using amino acid frequency; these correctly classify a large fraction of test cases with a high degree of specificity and sensitivity. Subsequently, we added estimates of protein isoelectric points as a feature to ANNs that classify specialized families of proteins, namely major capsid and tail proteins. As expected, these more specialized ANNs are more accurate than the structural ANNs. To experimentally validate the ANN predictions, several ORFs with no significant similarities to known sequences that are ANN-predicted structural proteins were examined by transmission electron microscopy. Some of these self-assembled into structures strongly resembling virion structures. Thus, our ANNs are new tools for identifying phage and potential prophage structural proteins that are difficult or impossible to detect by other bioinformatic analysis. The networks will be valuable when sequence is

  14. The agricultural antibiotic carbadox induces phage-mediated gene transfer in Salmonella

    PubMed Central

    Bearson, Bradley L.; Allen, Heather K.; Brunelle, Brian W.; Lee, In Soo; Casjens, Sherwood R.; Stanton, Thaddeus B.

    2013-01-01

    Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the US during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli (STEC) and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness genes in the

  15. Evolution of parasitism and mutualism between filamentous phage M13 and Escherichia coli

    PubMed Central

    Williams, Elizabeth S.C.P.; Turner, Paul E.

    2016-01-01

    Background. How host-symbiont interactions coevolve between mutualism and parasitism depends on the ecology of the system and on the genetic and physiological constraints of the organisms involved. Theory often predicts that greater reliance on horizontal transmission favors increased costs of infection and may result in more virulent parasites or less beneficial mutualists. We set out to understand transitions between parasitism and mutualism by evolving the filamentous bacteriophage M13 and its host Escherichia coli. Results. The effect of phage M13 on bacterial fitness depends on the growth environment, and initial assays revealed that infected bacteria reproduce faster and to higher density than uninfected bacteria in 96-well microplates. These data suggested that M13 is, in fact, a facultative mutualist of E. coli. We then allowed E. coli and M13 to evolve in replicated environments, which varied in the relative opportunity for horizontal and vertical transmission of phage in order to assess the evolutionary stability of this mutualism. After 20 experimental passages, infected bacteria from treatments with both vertical and horizontal transmission of phage had evolved the fastest growth rates. At the same time, phage from these treatments no longer benefited the ancestral bacteria. Conclusions. These data suggest a positive correlation between the positive effects of M13 on E. coli hosts from the same culture and the negative effects of the same phage toward the ancestral bacterial genotype. The results also expose flaws in applying concepts from the virulence-transmission tradeoff hypothesis to mutualism evolution. We discuss the data in the context of more recent theory on how horizontal transmission affects mutualisms and explore how these effects influence phages encoding virulence factors in pathogenic bacteria. PMID:27257543

  16. Artificial neural networks trained to detect viral and phage structural proteins.

    PubMed

    Seguritan, Victor; Alves, Nelson; Arnoult, Michael; Raymond, Amy; Lorimer, Don; Burgin, Alex B; Salamon, Peter; Segall, Anca M

    2012-01-01

    Phages play critical roles in the survival and pathogenicity of their hosts, via lysogenic conversion factors, and in nutrient redistribution, via cell lysis. Analyses of phage- and viral-encoded genes in environmental samples provide insights into the physiological impact of viruses on microbial communities and human health. However, phage ORFs are extremely diverse of which over 70% of them are dissimilar to any genes with annotated functions in GenBank. Better identification of viruses would also aid in better detection and diagnosis of disease, in vaccine development, and generally in better understanding the physiological potential of any environment. In contrast to enzymes, viral structural protein function can be much more challenging to detect from sequence data because of low sequence conservation, few known conserved catalytic sites or sequence domains, and relatively limited experimental data. We have designed a method of predicting phage structural protein sequences that uses Artificial Neural Networks (ANNs). First, we trained ANNs to classify viral structural proteins using amino acid frequency; these correctly classify a large fraction of test cases with a high degree of specificity and sensitivity. Subsequently, we added estimates of protein isoelectric points as a feature to ANNs that classify specialized families of proteins, namely major capsid and tail proteins. As expected, these more specialized ANNs are more accurate than the structural ANNs. To experimentally validate the ANN predictions, several ORFs with no significant similarities to known sequences that are ANN-predicted structural proteins were examined by transmission electron microscopy. Some of these self-assembled into structures strongly resembling virion structures. Thus, our ANNs are new tools for identifying phage and potential prophage structural proteins that are difficult or impossible to detect by other bioinformatic analysis. The networks will be valuable when sequence is

  17. Tolerance of a phage element by Streptococcus pneumoniae leads to a fitness defect during colonization.

    PubMed

    DeBardeleben, Hilary K; Lysenko, Elena S; Dalia, Ankur B; Weiser, Jeffrey N

    2014-07-01

    The pathogenesis of the disease caused by Streptococcus pneumoniae begins with colonization of the upper respiratory tract. Temperate phages have been identified in the genomes of up to 70% of clinical isolates. How these phages affect the bacterial host during colonization is unknown. Here, we examined a clinical isolate that carries a novel prophage element, designated Spn1, which was detected in both integrated and episomal forms. Surprisingly, both lytic and lysogenic Spn1 genes were expressed under routine growth conditions. Using a mouse model of asymptomatic colonization, we demonstrate that the Spn1(-) strain outcompeted the Spn1(+) strain >70-fold. To determine if Spn1 causes a fitness defect through a trans-acting factor, we constructed an Spn1(+) mutant that does not become an episome or express phage genes. This mutant competed equally with the Spn1(-) strain, indicating that expression of phage genes or phage lytic activity is required to confer this fitness defect. In vitro, we demonstrate that the presence of Spn1 correlated with a defect in LytA-mediated autolysis. Furthermore, the Spn1(+) strain displayed increased chain length and resistance to lysis by penicillin compared to the Spn(-) strain, indicating that Spn1 alters the cell wall physiology of its host strain. We posit that these changes in cell wall physiology allow for tolerance of phage gene products and are responsible for the relative defect of the Spn1(+) strain during colonization. This study provides new insight into how bacteria and prophages interact and affect bacterial fitness in vivo. PMID:24816604

  18. The phage growth limitation system in Streptomyces coelicolor A(3)2 is a toxin/antitoxin system, comprising enzymes with DNA methyltransferase, protein kinase and ATPase activity

    PubMed Central

    Hoskisson, Paul A.; Sumby, Paul; Smith, Margaret C.M.

    2015-01-01

    The phage growth limitation system of Streptomyces coelicolor A3(2) is an unusual bacteriophage defence mechanism. Progeny ϕC31 phage from an initial infection are thought to be modified such that subsequent infections are attenuated in a Pgl+ host but normal in a Pgl− strain. Earlier work identified four genes required for phage resistance by Pgl. Here we demonstrate that Pgl is an elaborate and novel phage restriction system that, in part, comprises a toxin/antitoxin system where PglX, a DNA methyltransferase is toxic in the absence of a functional PglZ. In addition, the ATPase activity of PglY and a protein kinase activity in PglW are shown to be essential for phage resistance by Pgl. We conclude that on infection of a Pgl+ cell by bacteriophage ϕC31, PglW transduces a signal, probably via phosphorylation, to other Pgl proteins resulting in the activation of the DNA methyltransferase, PglX and this leads to phage restriction. PMID:25592393

  19. Autophagy induction by SIRT6 through attenuation of insulin-like growth factor signaling is involved in the regulation of human bronchial epithelial cell senescence.

    PubMed

    Takasaka, Naoki; Araya, Jun; Hara, Hiromichi; Ito, Saburo; Kobayashi, Kenji; Kurita, Yusuke; Wakui, Hiroshi; Yoshii, Yutaka; Yumino, Yoko; Fujii, Satoko; Minagawa, Shunsuke; Tsurushige, Chikako; Kojima, Jun; Numata, Takanori; Shimizu, Kenichiro; Kawaishi, Makoto; Kaneko, Yumi; Kamiya, Noriki; Hirano, Jun; Odaka, Makoto; Morikawa, Toshiaki; Nishimura, Stephen L; Nakayama, Katsutoshi; Kuwano, Kazuyoshi

    2014-02-01

    Cigarette smoke (CS)-induced cellular senescence has been implicated in the pathogenesis of chronic obstructive pulmonary disease, and SIRT6, a histone deacetylase, antagonizes this senescence, presumably through the attenuation of insulin-like growth factor (IGF)-Akt signaling. Autophagy controls cellular senescence by eliminating damaged cellular components and is negatively regulated by IGF-Akt signaling through the mammalian target of rapamycin (mTOR). SIRT1, a representative sirtuin family, has been demonstrated to activate autophagy, but a role for SIRT6 in autophagy activation has not been shown. Therefore, we sought to investigate the regulatory role for SIRT6 in autophagy activation during CS-induced cellular senescence. SIRT6 expression levels were modulated by cDNA and small interfering RNA transfection in human bronchial epithelial cells (HBECs). Senescence-associated β-galactosidase staining and Western blotting of p21 were performed to evaluate senescence. We demonstrated that SIRT6 expression levels were decreased in lung homogenates from chronic obstructive pulmonary disease patients, and SIRT6 expression levels correlated significantly with the percentage of forced expiratory volume in 1 s/forced vital capacity. CS extract (CSE) suppressed SIRT6 expression in HBECs. CSE-induced HBEC senescence was inhibited by SIRT6 overexpression, whereas SIRT6 knockdown and mutant SIRT6 (H133Y) without histone deacetylase activity enhanced HBEC senescence. SIRT6 overexpression induced autophagy via attenuation of IGF-Akt-mTOR signaling. Conversely, SIRT6 knockdown and overexpression of a mutant SIRT6 (H133Y) inhibited autophagy. Autophagy inhibition by knockdown of ATG5 and LC3B attenuated the antisenescent effect of SIRT6 overexpression. These results suggest that SIRT6 is involved in CSE-induced HBEC senescence via autophagy regulation, which can be attributed to attenuation of IGF-Akt-mTOR signaling. PMID:24367027

  20. Properties and genomic analysis of Lactococcus garvieae lysogenic bacteriophage PLgT-1, a new member of Siphoviridae, with homology to Lactococcus lactis phages.

    PubMed

    Hoai, Truong Dinh; Nishiki, Issei; Yoshida, Terutoyo

    2016-08-15

    The lysogenic phage PLgT-1 is highly prevalent in Lactococcus garvieae, which is a serious bacterial pathogen in marine fish. Therefore, information regarding this phage is one of the key factors to predict the evolution of this bacterium. However, many properties of this phage, its complete genome sequence, and its relationship with other viral communities has not been investigated to date. Here, we demonstrated that the phage PLgT-1 was not only induced by an induction agent (Mitomycin C), but could be released frequently during cell division in a nutrient-rich environment or in natural seawater. Integration of PLgT-1 into non-lysogenic bacteria via transduction changed the genotype, resulting in the diversification of L. garvieae. The complete DNA sequence of PLgT-1 was also determined. This phage has a dsDNA genome of 40,273bp with 66 open reading frames (ORFs). Of these, the biological functions of 24 ORFs could be predicted but those of 42 ORFs are unknown. Thus, PLgT-1 is a novel phage with several novel proteins encoded in its genome. The strict MegaBLAST search program for the PLgT-1 genome revealed that this phage had no similarities with other previously investigated phages specific to L. garvieae (WP-2 and GE1). Notably, PLgT-1 was relatively homologous with several phages of Lactococcus lactis and 17 of the 24 predicted proteins encoded in PLgT-1 were homologous with the deduced proteins of various phages from these dairy bacteria. Comparative genome analysis revealed that the L. garvieae phage PLgT-1 was most closely related to the L. lactis phage TP712. However, they differed from each other in genome size and gene arrangement. The results obtained in this study suggest that the lysogenic phage PLgT-1 is a new member of the family Siphoviridae and has been involved in horizontal gene exchange with microbial communities, especially with L. lactis and its phages. PMID:27234995

  1. Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion

    PubMed Central

    Brüssow, Harald; Canchaya, Carlos; Hardt, Wolf-Dietrich

    2004-01-01

    Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like “swarms” of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework. PMID:15353570

  2. Efficacy of two Staphylococcus aureus phage cocktails in cheese production.

    PubMed

    El Haddad, Lynn; Roy, Jean-Pierre; Khalil, Georges E; St-Gelais, Daniel; Champagne, Claude P; Labrie, Steve; Moineau, Sylvain

    2016-01-18

    Staphylococcus aureus is one of the most prevalent pathogenic bacteria contaminating dairy products. In an effort to reduce food safety risks, virulent phages are investigated as antibacterial agents to control foodborne pathogens. The aim of this study was to compare sets of virulent phages, design phage cocktails, and use them in a cocktail to control pathogenic staphylococci in cheese. Six selected phages belonging to the three Caudovirales families (Myoviridae, Siphoviridae, Podoviridae) were strictly lytic, had a broad host range, and did not carry genes coding for virulence traits in their genomes. However, they were sensitive to pasteurization. At MOI levels of 15, 45, and 150, two anti-S. aureus phage cocktails, each containing three phages, one from each of the three phage families, eradicated a 10(6)CFU/g S. aureus population after 14 days of Cheddar cheese curd ripening at 4°C. The use of these phages did not trigger over-production of S. aureus enterotoxin C. The use of phage cocktails and their rotation may prevent the emergence of phage resistant bacterial strains. PMID:26476571

  3. Engineering resistance to phage GVE3 in Geobacillus thermoglucosidasius.

    PubMed

    van Zyl, Leonardo Joaquim; Taylor, Mark Paul; Trindade, Marla

    2016-02-01

    Geobacillus thermoglucosidasius is a promising platform organism for the production of biofuels and other metabolites of interest. G. thermoglucosidasius fermentations could be subject to bacteriophage-related failure and financial loss. We develop two strains resistant to a recently described G. thermoglucosidasius-infecting phage GVE3. The phage-encoded immunity gene, imm, was overexpressed in the host leading to phage resistance. A phage-resistant mutant was isolated following expression of a putative anti-repressor-like protein and phage challenge. A point mutation was identified in the polysaccharide pyruvyl transferase, csaB. A double crossover knockout mutation of csaB confirmed its role in the phage resistance phenotype. These resistance mechanisms appear to prevent phage DNA injection and/or lysogenic conversion rather than just reducing efficiency of plating, as no phage DNA could be detected in resistant bacteria challenged with GVE3 and no plaques observed even at high phage titers. Not only do the strains developed here shed light on the biological relationship between the GVE3 phage and its host, they could be employed by those looking to make use of this organism for metabolite production, with reduced occurrence of GVE3-related failure. PMID:26536875

  4. SN50, a Cell-Permeable Inhibitor of Nuclear Factor-κB, Attenuates Ventilator-Induced Lung Injury in an Isolated and Perfused Rat Lung Model.

    PubMed

    Chian, Chih-Feng; Chiang, Chi-Huei; Chuang, Chiao-Hui; Liu, Shiou-Ling; Tsai, Chen-Liang

    2016-08-01

    High tidal volume (VT) ventilation causes the release of various mediators and results in ventilator-induced lung injury (VILI). SN50, a cell-permeable nuclear factor-κB (NF-κB) inhibitory peptide, attenuates inflammation and acute respiratory distress syndrome. However, the mechanisms associated with the effects of SN50 in VILI have not been fully elucidated. We investigated the cellular and molecular mechanisms for the effects of SN50 treatment in VILI. An isolated and perfused rat lung model was exposed to low (5 mL/kg) or high (15 mL/kg) VT ventilation for 6 h. SN50 was administered in the perfusate at the onset of the high-stretch mechanical ventilation. The hemodynamics, lung histological changes, inflammatory responses, and activation of apoptotic pathways were evaluated. VILI was demonstrated by increased pulmonary vascular permeability and lung weight gain, as well as by increased levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, myeloperoxidase (MPO), hydrogen peroxide, and macrophage inflammatory protein-2 in the bronchoalveolar lavage fluid. The lung tissue expression of TNF-α, IL-1β, mitogen-activated protein kinases (MAPKs), caspase-3, and phosphorylation of serine/threonine-specific protein kinase (p-AKT) was greater in the high VT group than in the low VT group. Upregulation and activation of NF-κB was associated with increased lung injury in VILI. SN50 attenuated the inflammatory responses, including the expression of IL-1β, TNF-α, MPO, MAPKs, and NF-κB. In addition, the downregulation of apoptosis was evaluated using caspase-3 and p-AKT expression. Furthermore, SN50 mitigated the increases in the lung weights, pulmonary vascular permeability, and lung injury. In conclusion, VILI is associated with inflammatory responses and activation of NF-κB. SN50 inhibits the activation of NF-κB and attenuates VILI. PMID:26780513

  5. Precisely modulated pathogenicity island interference with late phage gene transcription.

    PubMed

    Ram, Geeta; Chen, John; Ross, Hope F; Novick, Richard P

    2014-10-01

    Having gone to great evolutionary lengths to develop resistance to bacteriophages, bacteria have come up with resistance mechanisms directed at every aspect of the bacteriophage life cycle. Most genes involved in phage resistance are carried by plasmids and other mobile genetic elements, including bacteriophages and their relatives. A very special case of phage resistance is exhibited by the highly mobile phage satellites, staphylococcal pathogenicity islands (SaPIs), which carry and disseminate superantigen and other virulence genes. Unlike the usual phage-resistance mechanisms, the SaPI-encoded interference mechanisms are carefully crafted to ensure that a phage-infected, SaPI-containing cell will lyse, releasing the requisite crop of SaPI particles as well as a greatly diminished crop of phage particles. Previously described SaPI interference genes target phage functions that are not required for SaPI particle production and release. Here we describe a SaPI-mediated interference system that affects expression of late phage gene transcription and consequently is required for SaPI and phage. Although when cloned separately, a single SaPI gene totally blocks phage production, its activity in situ is modulated accurately by a second gene, achieving the required level of interference. The advantage for the host bacteria is that the SaPIs curb excessive phage growth while enhancing their gene transfer activity. This activity is in contrast to that of the clustered regularly interspaced short palindromic repeats (CRISPRs), which totally block phage growth at the cost of phage-mediated gene transfer. In staphylococci the SaPI strategy seems to have prevailed during evolution: The great majority of Staphylococcus aureus strains carry one or more SaPIs, whereas CRISPRs are extremely rare. PMID:25246539

  6. Aryl Hydrocarbon Receptor Antagonism Attenuates Growth Factor Expression, Proliferation, and Migration in Fibroblast-Like Synoviocytes from Patients with Rheumatoid Arthritis

    PubMed Central

    Lahoti, Tejas S.; Hughes, Jarod M.; Kusnadi, Ann; John, Kaarthik; Zhu, Bokai; Murray, Iain A.; Gowda, Krishne; Peters, Jeffrey M.; Amin, Shantu G.

    2014-01-01

    Rheumatoid arthritis (RA) is a chronic autoimmune disease with high morbidity and mortality. Within the inflammatory milieu, resident fibroblast-like synoviocytes (FLS) in the synovial tissue undergo hyperplasia, which leads to joint destruction. Epidemiologic studies and our previous research suggest that activation of the aryl hydrocarbon receptor (AHR) pathway plays an instrumental role in the inflammatory and destructive RA phenotype. In addition, our recent studies implicate the AHR in the regulation of the expression of several growth factors in established tumor cell lines. Thus, under inflammatory conditions, we hypothesized that the AHR is involved in the constitutive and inducible expression of several growth factors, FLS proliferation and migration, along with protease-dependent invasion in FLS from patients with RA (RA-FLS). Treatment with the AHR antagonist GNF351 inhibits cytokine-induced expression of vascular endothelial growth factor-A (VEGF-A), epiregulin, amphiregulin, and basic fibroblast growth factor mRNA through an AHR-dependent mechanism in both RA-FLS and FLS. Secretion of VEGF-A and epiregulin from RA-FLS was also inhibited upon GNF351 treatment. RA-FLS cell migration, along with cytokine-induced RA-FLS cell proliferation, was significantly attenuated by GNF351 exposure. Treatment of RA-FLS with GNF351 mitigated cytokine-mediated expression of matrix metalloproteinase-2 and -9 mRNA and diminished the RA-FLS invasive phenotype. These findings indicate that inhibition of AHR activity may be a viable therapeutic target in amelioration of disease progression in RA by attenuating growth factor release; FLS proliferation, migration, and invasion; and inflammatory activity. PMID:24309559

  7. An early granulocyte colony-stimulating factor treatment attenuates neuropathic pain through activation of mu opioid receptors on the injured nerve

    PubMed Central

    Liao, Ming-Feng; Yeh, Shin-Rung; Lo, Ai-Lun; Chao, Po-Kuan; Lee, Yun-Lin; Hung, Yu-Hui; Lu, Kwok-Tung; Ro, Long-Sun

    2016-01-01

    Several studies have shown that the mu opioid receptor (MOR) located in the peripheral nerves can be activated after nerve injury and that it attenuates peripheral nociceptive signals to the spinal dorsal horn. Various cytokines and phosphorylated-p38 (p-p38) activation in the dorsal horn also play an important role in neuropathic pain development. Granulocyte-colony stimulating factor (GCSF) is a growth factor that can stimulate granulocyte formation and has been shown to exert an analgesic effect on neuropathic pain through recruiting opioid-containing leukocytes to the injured nerve. However, the underlying mechanisms are not well understood. Herein, the results of behavior tests in addition to MOR levels in the injured sciatic nerve and the levels of p-p38 and various cytokines in the spinal dorsal horn were studied in vehicle-treated or GCSF-treated chronic constriction injured (CCI) rats at different time points (i.e., 1, 3, and 7 days, respectively) after nerve injury. The results showed that a single early systemic GCSF treatment after nerve injury can up-regulate MORs in the injured nerve, which can decrease peripheral nociceptive signals. Thereafter, those changes suppress the pro-inflammatory cytokine IL-6 but enhance the anti-inflammatory cytokine IL-4, followed by decreases in p-p38 in the dorsal horn, and thus further attenuate neuropathic pain. PMID:27180600

  8. An early granulocyte colony-stimulating factor treatment attenuates neuropathic pain through activation of mu opioid receptors on the injured nerve.

    PubMed

    Liao, Ming-Feng; Yeh, Shin-Rung; Lo, Ai-Lun; Chao, Po-Kuan; Lee, Yun-Lin; Hung, Yu-Hui; Lu, Kwok-Tung; Ro, Long-Sun

    2016-01-01

    Several studies have shown that the mu opioid receptor (MOR) located in the peripheral nerves can be activated after nerve injury and that it attenuates peripheral nociceptive signals to the spinal dorsal horn. Various cytokines and phosphorylated-p38 (p-p38) activation in the dorsal horn also play an important role in neuropathic pain development. Granulocyte-colony stimulating factor (GCSF) is a growth factor that can stimulate granulocyte formation and has been shown to exert an analgesic effect on neuropathic pain through recruiting opioid-containing leukocytes to the injured nerve. However, the underlying mechanisms are not well understood. Herein, the results of behavior tests in addition to MOR levels in the injured sciatic nerve and the levels of p-p38 and various cytokines in the spinal dorsal horn were studied in vehicle-treated or GCSF-treated chronic constriction injured (CCI) rats at different time points (i.e., 1, 3, and 7 days, respectively) after nerve injury. The results showed that a single early systemic GCSF treatment after nerve injury can up-regulate MORs in the injured nerve, which can decrease peripheral nociceptive signals. Thereafter, those changes suppress the pro-inflammatory cytokine IL-6 but enhance the anti-inflammatory cytokine IL-4, followed by decreases in p-p38 in the dorsal horn, and thus further attenuate neuropathic pain. PMID:27180600

  9. Indirect Ultraviolet-Reactivation of Phage λ

    PubMed Central

    George, Jacqueline; Devoret, Raymond; Radman, Miroslav

    1974-01-01

    When an F- recipient Escherichia coli K12 bacterium receives Hfr or F-lac+ DNA from an ultraviolet-irradiated donor, its capacity to promote DNA repair and mutagenesis of ultraviolet-damaged phage λ is substantially increased. We call this phenomenon indirect ultraviolet-reactivation, since its features are essentially the same as those of ultraviolet-reactivation; this repair process occurs in pyrimidine dimer excision-deficient strains and produces clear plaque mutations of the restored phage. Moreover, this process is similar to indirect ultraviolet-induction of prophage λ, since it is promoted by conjugation. However, contrarily to indirect induction, it is produced by Hfr donors and occurs in recipients restricting the incoming ultraviolet-damaged donor DNA. The occurrence of indirect ultraviolet-reactivation provides evidence for the existence in E. coli of an inducible error-prone mechanism for the repair of DNA. PMID:4589889

  10. Targeted bacterial immunity buffers phage diversity.

    PubMed

    Haerter, Jan O; Trusina, Ala; Sneppen, Kim

    2011-10-01

    Bacteria have evolved diverse defense mechanisms that allow them to fight viral attacks. One such mechanism, the clustered, regularly interspaced, short palindromic repeat (CRISPR) system, is an adaptive immune system consisting of genetic loci that can take up genetic material from invasive elements (viruses and plasmids) and later use them to reject the returning invaders. It remains an open question how, despite the ongoing evolution of attack and defense mechanisms, bacteria and viral phages manage to coexist. Using a simple mathematical model and a two-dimensional numerical simulation, we found that CRISPR adaptive immunity allows for robust phage-bacterium coexistence even when the number of virus species far exceeds the capacity of CRISPR-encoded genetic memory. Coexistence is predicted to be a consequence of the presence of many interdependent species that stress but do not overrun the bacterial defense system. PMID:21813617