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Sample records for attenuating factor phage

  1. Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

    PubMed Central

    Filippov, Andrey A.; Sergueev, Kirill V.; He, Yunxiu; Huang, Xiao-Zhe; Gnade, Bryan T.; Mueller, Allen J.; Fernandez-Prada, Carmen M.; Nikolich, Mikeljon P.

    2011-01-01

    Background Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. Methodology/Principal Findings The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. Conclusions/Significance We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis. PMID:21980477

  2. Phages can constrain protist predation-driven attenuation of Pseudomonas aeruginosa virulence in multienemy communities

    PubMed Central

    Friman, Ville-Petri; Buckling, Angus

    2014-01-01

    The coincidental theory of virulence predicts that bacterial pathogenicity could be a by-product of selection by natural enemies in environmental reservoirs. However, current results are ambiguous and the simultaneous impact of multiple ubiquitous enemies, protists and phages on virulence evolution has not been investigated previously. Here we tested experimentally how Tetrahymena thermophila protist predation and PNM phage parasitism (bacteria-specific virus) alone and together affect the evolution of Pseudomonas aeruginosa PAO1 virulence, measured in wax moth larvae. Protist predation selected for small colony types, both in the absence and presence of phage, which showed decreased edibility to protists, reduced growth in the absence of enemies and attenuated virulence. Although phage selection alone did not affect the bacterial phenotype, it weakened protist-driven antipredatory defence (biofilm formation), its associated pleiotropic growth cost and the correlated reduction in virulence. These results suggest that protist selection can be a strong coincidental driver of attenuated bacterial virulence, and that phages can constrain this effect owing to effects on population dynamics and conflicting selection pressures. Attempting to define causal links such as these might help us to predict the cold and hot spots of coincidental virulence evolution on the basis of microbial community composition of environmental reservoirs. PMID:24671085

  3. Phages can constrain protist predation-driven attenuation of Pseudomonas aeruginosa virulence in multienemy communities.

    PubMed

    Friman, Ville-Petri; Buckling, Angus

    2014-09-01

    The coincidental theory of virulence predicts that bacterial pathogenicity could be a by-product of selection by natural enemies in environmental reservoirs. However, current results are ambiguous and the simultaneous impact of multiple ubiquitous enemies, protists and phages on virulence evolution has not been investigated previously. Here we tested experimentally how Tetrahymena thermophila protist predation and PNM phage parasitism (bacteria-specific virus) alone and together affect the evolution of Pseudomonas aeruginosa PAO1 virulence, measured in wax moth larvae. Protist predation selected for small colony types, both in the absence and presence of phage, which showed decreased edibility to protists, reduced growth in the absence of enemies and attenuated virulence. Although phage selection alone did not affect the bacterial phenotype, it weakened protist-driven antipredatory defence (biofilm formation), its associated pleiotropic growth cost and the correlated reduction in virulence. These results suggest that protist selection can be a strong coincidental driver of attenuated bacterial virulence, and that phages can constrain this effect owing to effects on population dynamics and conflicting selection pressures. Attempting to define causal links such as these might help us to predict the cold and hot spots of coincidental virulence evolution on the basis of microbial community composition of environmental reservoirs.

  4. The factors affecting effectiveness of treatment in phages therapy

    PubMed Central

    Ly-Chatain, Mai Huong

    2014-01-01

    In recent years, the use of lytic bacteriophages as antimicrobial agents controlling pathogenic bacteria has appeared as a promising new alternative strategy in the face of growing antibiotic resistance which has caused problems in many fields including medicine, veterinary medicine, and aquaculture. The use of bacteriophages has numerous advantages over traditional antimicrobials. The effectiveness of phage applications in fighting against pathogenic bacteria depends on several factors such as the bacteriophages/target bacteria ratio, the mode and moment of treatment, environmental conditions (pH, temperature...), the neutralization of phage and accessibility to target bacteria, amongst others. This report presents these factors and the challenges involved in developing phage therapy applications. PMID:24600439

  5. 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

  6. 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.

  7. The genomes, proteomes, and structures of three novel phages that infect the Bacillus cereus group and carry putative virulence factors.

    PubMed

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

    2014-10-01

    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 the

  8. 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

  9. 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.

  10. 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

  11. 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).

  12. Phage Displayed Peptides/Antibodies Recognizing Growth Factors and Their Tyrosine Kinase Receptors as Tools for Anti-Cancer Therapeutics

    PubMed Central

    Ronca, Roberto; Benzoni, Patrizia; De Luca, Angela; Crescini, Elisabetta; Dell’Era, Patrizia

    2012-01-01

    The basic idea of displaying peptides on a phage, introduced by George P. Smith in 1985, was greatly developed and improved by McCafferty and colleagues at the MRC Laboratory of Molecular Biology and, later, by Barbas and colleagues at the Scripps Research Institute. Their approach was dedicated to building a system for the production of antibodies, similar to a naïve B cell repertoire, in order to by-pass the standard hybridoma technology that requires animal immunization. Both groups merged the phage display technology with an antibody library to obtain a huge number of phage variants, each of them carrying a specific antibody ready to bind its target molecule, allowing, later on, rare phage (one in a million) to be isolated by affinity chromatography. Here, we will briefly review the basis of the technology and the therapeutic application of phage-derived bioactive molecules when addressed against key players in tumor development and progression: growth factors and their tyrosine kinase receptors. PMID:22606042

  13. 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

  14. 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.

  15. Factors Affecting Phage D29 Infection: A Tool to Investigate Different Growth States of Mycobacteria

    PubMed Central

    Swift, Benjamin M. C.; Gerrard, Zara E.; Huxley, Jonathan N.; Rees, Catherine E. D.

    2014-01-01

    Bacteriophages D29 and TM4 are able to infect a wide range of mycobacteria, including pathogenic and non-pathogenic species. Successful phage infection of both fast- and slow-growing mycobacteria can be rapidly detected using the phage amplification assay. Using this method, the effect of oxygen limitation during culture of mycobacteria on the success of phage infection was studied. Both D29 and TM4 were able to infect cultures of M. smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) grown in liquid with aeration. However when cultures were grown under oxygen limiting conditions, only TM4 could productively infect the cells. Cell attachment assays showed that D29 could bind to the cells surface but did not complete the lytic cycle. The ability of D29 to productively infect the cells was rapidly recovered (within 1 day) when the cultures were returned to an aerobic environment and this recovery required de novo RNA synthesis. These results indicated that under oxygen limiting conditions the cells are entering a growth state which inhibits phage D29 replication, and this change in host cell biology which can be detected by using both phage D29 and TM4 in the phage amplification assay. PMID:25184428

  16. Soil temperature effect in calculating attenuation and retardation factors.

    PubMed

    Paraiba, Lourival Costa; Spadotto, Claudio Aparecido

    2002-09-01

    The effect of annual variation of daily average soil temperature, at different depths, in calculating pesticides ranking indexes retardation factor and attenuation factor is presented. The retardation factor and attenuation factor are two site-specific pesticide numbers, frequently used as screening indicator indexes for pesticide groundwater contamination potential. Generally, in the calculation of these two factors are not included the soil temperature effect on the parameters involved in its calculation. It is well known that the soil temperature affects the pesticide degradation rate, water-air partition coefficient and water-soil partition coefficient. These three parameters are components of the retardation factor and attenuation factor and contribute to determine the pesticide behavior in the environment. The Arrhenius equation, van't Hoff equation and Clausius-Clapeyron equation are used in this work for estimating the soil temperature effect on the pesticide degradation rate, water-air partition coefficient and soil-water partition coefficient, respectively. These dependence relationships, between results of calculating attenuation and retardation factors and the soil temperature at different depths, can aid to understand the potential pesticide groundwater contamination on different weather conditions. Numerical results will be presented with pesticides atrazine and lindane in a soil profile with 20 degrees C constant temperature, minimum and maximum surface temperatures varying and spreading in the soil profile between -5 and 30 degrees C and between 15 and 45 degrees C.

  17. 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

  18. 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.

  19. 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

  20. Structure of a group A streptococcal phage-encoded virulence factor reveals a catalytically active triple-stranded beta-helix.

    PubMed

    Smith, Nicola L; Taylor, Edward J; Lindsay, Anna-Marie; Charnock, Simon J; Turkenburg, Johan P; Dodson, Eleanor J; Davies, Gideon J; Black, Gary W

    2005-12-01

    Streptococcus pyogenes (group A Streptococcus) causes severe invasive infections including scarlet fever, pharyngitis (streptococcal sore throat), skin infections, necrotizing fasciitis (flesh-eating disease), septicemia, erysipelas, cellulitis, acute rheumatic fever, and toxic shock. The conversion from nonpathogenic to toxigenic strains of S. pyogenes is frequently mediated by bacteriophage infection. One of the key bacteriophage-encoded virulence factors is a putative "hyaluronidase," HylP1, a phage tail-fiber protein responsible for the digestion of the S. pyogenes hyaluronan capsule during phage infection. Here we demonstrate that HylP1 is a hyaluronate lyase. The 3D structure, at 1.8-angstroms resolution, reveals an unusual triple-stranded beta-helical structure and provides insight into the structural basis for phage tail assembly and the role of phage tail proteins in virulence. Unlike the triple-stranded beta-helix assemblies of the bacteriophage T4 injection machinery and the tailspike endosialidase of the Escherichia coli K1 bacteriophage K1F, HylP1 possesses three copies of the active center on the triple-helical fiber itself without the need for an accessory catalytic domain. The triple-stranded beta-helix is not simply a structural scaffold, as previously envisaged; it is harnessed to provide a 200-angstroms-long substrate-binding groove for the optimal reduction in hyaluronan viscosity to aid phage penetration of the capsule.

  1. 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.

  2. P7, a novel antagonist of corticotropin releasing factor receptor type 1 (CRFR1) screened from phage display library.

    PubMed

    Yu, Jinmei; Zhuo, Rengong; Peng, Peng; Liu, Xiaoyan; Yan, Haitao; Zhang, Shuzhuo; Zheng, Jianquan; Wei, Xiaoli; Ma, Xiaoyun

    2015-07-31

    The corticotropin releasing factor (CRF) plays a central role in regulating the activities of hypothalamic-pituitary-adrenal (HPA) axis in the presence of a variety of stressful stimuli via binding to its type 1 receptors (CRFR1). Despite that many peptidic or non-peptidic antagonists of CRFR1 have been developed to serve as therapeutic tools to CRF-related pathologies, none of them have been utilized clinically. Targeting the extracellular domain 1 (EC1) of CRFR1, the CRF-binding site, represents a new strategy to inhibit the function of the receptor. However, no such agents have been identified up to now. Herein, by using an 87-amino acid fragment corresponding to the EC1 region as the bait, we screened the binding polypeptides from a phage display (Ph.D.-12) peptide library. After 3-round biopanning, positive clones were selected and the polypeptides carried by them were identified. 5 polypeptides were found to bind with the target specifically. Among them, the P7 exhibited the highest affinity. By evaluating the cAMP accumulation in the CRFR1 or CRFR2-expressing HEK293 cells, we demonstrated that P7 blocking the function of CRFR1, but not CRFR2. In addition, we also found that P7 and CRF act on CRFR1 competitively. Taken together, we reveal that P7, a novel polypeptide identified from phage display library, inhibits the function of CRFR1 effectively and specifically by binding at its EC1 domain. The new polypeptide might provide a promising agent for diagnostic or therapeutic utilities in CRF-related disorders.

  3. Phage therapy pharmacology: calculating phage dosing.

    PubMed

    Abedon, Stephen

    2011-01-01

    Phage therapy, which can be described as a phage-mediated biocontrol of bacteria (or, simply, biocontrol), is the application of bacterial viruses-also bacteriophages or phages-to reduce densities of nuisance or pathogenic bacteria. Predictive calculations for phage therapy dosing should be useful toward rational development of therapeutic as well as biocontrol products. Here, I consider the theoretical basis of a number of concepts relevant to phage dosing for phage therapy including minimum inhibitory concentration (but also "inundation threshold"), minimum bactericidal concentration (but also "clearance threshold"), decimal reduction time (D value), time until bacterial eradication, threshold bacterial density necessary to support phage population growth ("proliferation threshold"), and bacterial density supporting half-maximal phage population growth rates (K(B)). I also address the concepts of phage killing titers, multiplicity of infection, and phage peak densities. Though many of the presented ideas are not unique to this chapter, I nonetheless provide variations on derivations and resulting formulae, plus as appropriate discuss relative importance. The overriding goal is to present a variety of calculations that are useful toward phage therapy dosing so that they may be found in one location and presented in a manner that allows facile appreciation, comparison, and implementation. The importance of phage density as a key determinant of the phage potential to eradicate bacterial targets is stressed throughout the chapter. PMID:22050820

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. Insights into aureocin A70 regulation: participation of regulator AurR, alternative transcription factor σ(B) and phage ϕ11 regulator cI.

    PubMed

    Coelho, Marcus Lívio Varella; Fleming, Luana Rocha; Bastos, Maria do Carmo de Freire

    2016-01-01

    Aureocin A70 is a four-component bacteriocin produced by Staphylococcus aureus A70. Its locus encompasses three transcriptional units coding for: (i) structural peptides (aurABCD), (ii) an ABC transporter (aurT) and (iii) the dedicated immunity protein and a putative transcriptional regulator (aurRI). The data provided here showed that AurR is an HTH-containing protein that reduces aureocin A70 production on solid medium, but not in broth. AurR seems to work similarly to LtnR and CylR2, repressors of lantibiotics lacticin 3147 and cytolysin, respectively. At least two other factors play a role in aureocin A70 production: (i) the alternative σ(B) factor, as σ(B)-defective cells produce more bacteriocin than the restored σ(B+) cells, and (ii) the ϕ11 regulator cI, since a lysogenic strain for ϕ11 exhibited a significant reduction in aureocin A70 production on solid medium when compared with the non-lysogenic isogenic strain. Full aeration and ROS generation abolished the effect of the phage regulators on aureocin A70 production. Interestingly, the ϕ11 regulator cI seems to cooperate with AurR to abolish aureocin A70 production. This study therefore represents the first report showing that phage regulators may play a role in regulation of bacteriocin production.

  13. Soil and groundwater attenuation factors for nitrogen from septic systems in the Chesapeake Bay TMDL

    NASA Astrophysics Data System (ADS)

    Radcliffe, D. E.; Geza, M.; O'Drisoll, M.; Humphrey, C., Jr.

    2015-12-01

    An expert panel was tasked with estimating the percent of the nitrogen (N) load from septic systems that was lost in the flow path from a typical home to third-order streams as part of the Chesapeake Bay Total Maximum Daily Load (TMDL). These losses were referred to as attenuation factors. We developed values for the soil (unsaturated) zone and for the Piedmont and Coastal Plain groundwater zones. For the soil zone, we used the Soil Treatment Unit MODel (STUMOD) to estimate loses due to denitrification for all 12 soil textural classes and then averaged the results over three textural groups. Assuming hydraulic loading at the design rate and a conventional system, the attenuation factors were 16% for sand, loamy sand, sandy loam, and loam soils; 34% for silt loam, clay loam, sandy clay loam, silty clay loam, and silt soils; and 54% for sandy clay, silty clay, and clay soils. Attenuation factors increased in the more clayey soils due to wetter conditions and more losses due to denitrification. Attenuation factors were also developed for reduced hydraulic loading rates and for systems using advanced N pre-treatment. For the Piedmont groundwater zone, we used data from a recent study in Georgia of small suburban streams with high-density septic systems. Stream base-flow load was estimated using simultaneous measurements of total N concentration and discharge and compared to the estimated groundwater input load, resulting in an attenuation factor of 81%. For the Coastal Plain groundwater zone, literature values of groundwater N concentrations within septic system plumes in Virginia, North Carolina, and Florida were used to estimate an attenuation factor of approximately 60% at 100m downgradient from the drainfield. These attenuation factors will be used to estimate the contribution of N to the Chesapeake Bay in the Phase 6 TMDL models.

  14. [Search for destruction factors of bacterial biofilms: comparison of phage properties in a group of Pseudomonas putida bacteriophages and specificity of their halo-formation products].

    PubMed

    Shaburova, O V; Krylov, S V; Veĭko, V P; Pleteneva, E A; Burkal'tseva, M V; Miroshnokov, K A; Kornelissen, A; Lavogne, R; Sykilinda, N N; Kadykov, V A; Mesianzhinov, V V; Volckaert, G; Krylov, V N

    2009-02-01

    Comparison of Pseudomonas putida group of phages attributed to five species (af, phi15, phi27, phi2F, and pf16) with their common property of halo-formation (formation of lightening zones) around phage plaques was conducted. The halo around phage plaques appears as a result of reduction or disappearance of bacterial polysaccharide capsules. The concentration of viable bacteria remains unchanged within the halo. A comparison of specificities of halo-formation products from various phages was conducted by a simple method. These products were shown to be highly specific and inactive on other species of pseudomonads. Phage-resistant P. putida mutants scored with respect to various phages, which lost phage adsorption ability, were tolerant to the effect of halo-formation products in most cases. Apparently, the capsular polysaccharides, which serve as a substrate for depolymerases and are the primary phage receptors, may be often lost. Results of partial sequencing of the af phage genome revealed an open reading frame that encodes the enzyme transglycosylase similar rather to transglycosylases of oligotrophic bacteria belonging to different species than to lysozymes of other phages. Possibly, it is a polyfunctional enzyme combining functions of lysozyme and an enzyme that executes the penetration of phage particle across extracellular slime and capsule. PMID:19334612

  15. Factors influencing the stability of live oral attenuated bacterial vaccines.

    PubMed

    Cryz, S J; Pasteris, O; Varallyay, S J; Fürer, E

    1996-01-01

    Live oral attenuated vaccines against typhoid fever (Salmonella typhi Ty21a) and cholera (Vibrio cholerae CVD 103-HgR) have been licensed for human use. Vaccine potency is dependent upon each dose containing a minimum number of viable organisms and galenic parameters. To ensure long-term stability, such vaccines must be stored at 5 degrees C +/- 3 degrees C. However, exposure to ambient temperatures (25 degrees C) for short periods of time (< 7 days) does not compromise vaccine potency. Brief exposures (< or = 24 hours) to temperatures as high as 37 degrees C will also not render the vaccine unsuitable for use. The Ty21a vaccine is available either as enteric-coated capsules or as a "liquid formulation", while CVD 103-HgR is presented only in the latter form. Each galenic formulation presents unique challenges with regard to the production of stable vaccines. Residual moisture, excipients, and processing temperatures during manufacturing were all found to markedly affect vaccine stability.

  16. Preparation and assay of phage lambda.

    PubMed

    Dale, J W; Greenaway, P J

    1985-01-01

    Lambda, a temperate bacteriophage of E. coli, has two alternative modes of replication in sensitive cells, known as the lytic and lysogenic cycles. In the lytic cycle, after the lambda DNA enters the cells, various phage functions are expressed that result in the production of a large number of mature phage particles and cell lysis. In the lysogenic mode, which normally occurs in only a small proportion of the infected cells, the phage forms a more or less stable relationship with the host bacterium; this stable state is known as lysogeny. In a lysogenic cell, phage DNA is normally incorporated into the chromosomal DNA via specific attachment sites on both the phage DNA and the host chromosome. Replication of lambda DNA then occurs only during replication of the host chromosome, and the phage genome is inherited by each daughter cell at cell division. The phage is maintained in this prophage state through the action of a repressor protein, coded for by the phage gene cl. This repressor protein turns off the expression of virtually the whole of the lambda genome. If the repressor is inactivated, the expression of phage genes is initiated. This leads to the excision of lambda DNA from the host chromosome and entry into the lytic cycle. The balance between the lytic and lysogenic modes of replication is a delicate and complex one in which a key factor is the concentration of the cl gene product. Some of the many sources of further information about the basic biology of lambda phage are listed in the references to this chapter.

  17. 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.

  18. N15: the linear phage-plasmid.

    PubMed

    Ravin, Nikolai V

    2011-03-01

    The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into chromosome but is a linear plasmid molecule with covalently closed ends. Upon infection the phage DNA circularises via cohesive ends, then phage-encoded enzyme, protelomerase, cuts at an inverted repeat site and forms hairpin ends (telomeres) of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally resulting in formation of duplicated telomeres. Then the N15 protelomerase cuts duplicated telomeres generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by partitioning operon similar to the F factor sop operon. Unlike F sop, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in N15 genome regions involved in phage replication and control of lysogeny, and binding of partition proteins at these sites regulates these processes. Two N15-related lambdoid Siphoviridae phages, φKO2 in Klebsiella oxytoca and pY54 in Yersinia enterocolitica, also lysogenize their hosts as linear plasmids, as well as Myoviridae marine phages VP882 and VP58.5 in Vibrio parahaemolyticus and ΦHAP-1 in Halomonas aquamarina. The genomes of all these phages contain similar protelomerase genes, lysogeny modules and replication genes, as well as plasmid-partitioning genes, suggesting that these phages may belong to a group diverged from a common ancestor.

  19. 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

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

    PubMed

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

    2015-04-01

    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 (c(in)) on subslab concentration (c(ss)) for residences in EPA database, an analytical equation is obtained to identify the relationship among c(in), css and the averaged background level. The new relationship indicates that subslab measurements may serve as a useful guide only if c(ss) is above 500 μg/m(3). Otherwise, c(in) is independent of c(ss), with a distribution in good agreements with other studies of background levels. Therefore, employing this screening value (500 μg/m(3)), 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 c(in) 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 c(in).

  1. Potential attenuation of p38 signaling by DDB2 as a factor in acquired TNF resistance.

    PubMed

    Sun, Chun-Ling; Chao, Chuck C-K

    2005-06-20

    Our previous study demonstrated that DDB2, a DNA repair protein, attenuates cell surface membrane-associated death signal induced by UV or FasAb; DDB2 is overexpressed in cisplatin-selected cells. However, the molecular mechanism underlying the protective role of DDB2 along the apoptotic pathway remains unknown. Our study identified the cross-resistance of the cisplatin-selected cells to tumor necrosis factor-alpha (TNF-alpha). Since knock-down of the DDB2 level rendered cells (HR18) sensitive to the treatment, the cell sensitivity to TNF-alpha appears inversely proportional to the cellular level of DDB2. Treatment of HeLa cells with TNF-alpha transiently induced activation of p38MAPK signal, but this induction was significantly reduced in the resistant cells. Overexpression of DDB2 attenuated the activation of p38 in cells. TNF-alpha-induced apoptotic signals, represented by caspase-8 and downstream substrate cleavage, were reduced in resistant cells compared to their sensitive counterparts. Inhibition of p38 signal by SB202190 clearly attenuated TNF-alpha-induced apoptotic signals. Moreover, overexpression of DDB2 in HR18 cells also attenuated TNF-alpha induced caspase activation. These results suggest that p38MAPK activation may be a key upstream signal of TNF-alpha-induced apoptosis and that attenuation of p38 signal by DDB2 overexpression may be responsible for acquired TNF-alpha resistance. PMID:15700318

  2. 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...

  3. 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

  4. 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

  5. Factors influencing the attenuation of serotype 1 Marek's disease virus by serial cell culture passage, and evaluation of attenuated strains for protection and replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was to better understand factors that influenced the process of attenuation of Marek’s disease (MD) virus by serial passage in cell cultures. Three virulent (v) pathotype and 3 very virulent plus (vv+) pathotype strains were passed by 3 techniques up to 131 times and the passage level at...

  6. 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

  7. 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

  8. 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.

  9. 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

  10. Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy

    PubMed Central

    Khan Mirzaei, Mohammadali; Nilsson, Anders S.

    2015-01-01

    Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection. PMID:25761060

  11. Paradoxical effects of the phage display-derived peptide antagonist IGF-F1-1 on insulin-like growth factor-1 receptor signaling.

    PubMed

    Robinson, Stephanie A; Rosenzweig, Steven A

    2006-06-28

    The insulin-like growth factor binding proteins (IGFBPs) represent a unique class of IGF antagonists regulating the bioavailability of the IGFs extracellularly. Accordingly, they represent an important class of proteins for cancer therapeutics and chemoprevention. IGF-F1-1 is a cyclic hexadecapeptide identified by high throughput phage display that binds to the IGFBP-binding domain on IGF-1. It acts as an IGFBP-mimetic, capable of inhibiting IGF-1 binding to the IGFBPs. To further examine the utility of IGF-F1-1 as an IGF-1 antagonist we tested its ability to inhibit IGFBP-2 and IGFBP-3 binding to IGF-1, (125)I-IGF-1 binding to IGF-1Rs and to block IGF-1 induced Akt activation, cell cycle changes and [(3)H]thymidine incorporation in MCF-7 cells. These biological activities were inhibited by treatment with IGFBP-2, wortmannin or the IGF-1R tyrosine kinase inhibitor, NVP-AEW541, but not by IGF-F1-1. Our findings confirm previous studies indicating that IGF-F1-1 is a weak antagonist of IGF-1 binding to the IGFBPs and the IGF-1R and suggest that it does not effectively inhibit downstream events stimulated by IGF-1. We further demonstrated that IGF-F1-1 treatment of MCF-7 cells results in the paradoxical activation of Akt, S-phase transition and [(3)H]thymidine incorporation. These results suggest that IGF-F1-1 is a weak agonist, exhibiting mitogenic actions. IGF-F1-1 may act in conjunction with IGF-1 at the IGF-1R or independently of IGF-1 at the IGF-1R or another receptor.

  12. 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.

  13. The first phage electron micrographs

    PubMed Central

    Ackermann, Hans-W.

    2011-01-01

    The first phage electron micrographs were published in 1940 in Germany and proved the particulate nature of bacteriophages. Phages and infected bacteria were first examined raw and unstained. US American scientists introduced shadowing and freeze-drying. Phages appeared to be tailed and morphologically heterogeneous. Phage types identified by early electron microscopy include enterobacteriophages T4, T1, T7, T5, 7–11, ViI and Pseudomonas phage PB1. This paper retraces the development of early virus electron microscopy till the introduction of negative staining. PMID:23050215

  14. 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.

  15. Insulin-like growth factor 1 treatment of MSCs attenuates inflammation and cardiac dysfunction following MI.

    PubMed

    Guo, Jun; Zheng, Dong; Li, Wen-feng; Li, Hai-rui; Zhang, Ai-dong; Li, Zi-cheng

    2014-12-01

    It has been reported that insulin-like growth factor 1 (IGF-1) promoted migration of endothelial cells and cardiac resident progenitor cells. In the previous study, we found the time-dependent and dose-dependent effects of IGF-1 treatment on the CXCR4 expression in MSCs in vitro, but it is still not clear whether IGF-1 pretreatment of MSCs may play anti-apoptotic and anti-inflammation role in myocardial infarction. In this study, we demonstrated that IGF-1-treated MSCs' transplantation attenuate cardiac dysfunction, increase the survival of engrafted cells in the ischemic heart, decrease myocardium cells apoptosis, and inhibit protein production and gene expression of inflammation cytokines tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6. IGF-1 pretreatment of MSCs may play anti-apoptotic and anti-inflammation roles in post-myocardial infarction.

  16. 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

  17. Replication and Maintenance of Linear Phage-Plasmid N15.

    PubMed

    Ravin, Nikolai V

    2015-02-01

    The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into the chromosome but is a linear plasmid molecule with covalently closed ends (telomeres). Upon infection, the phage DNA circularizes via cohesive ends, and then a special phage enzyme of the tyrosine recombinase family, protelomerase, cuts at another site and joins the ends, forming hairpin telomeres of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally, resulting in the formation of duplicated telomeres. The N15 protelomerase cuts them, generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by a partitioning operon similar to the F factor sop operon. Unlike the F centromere, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in the N15 genome regions involved in phage replication and control of lytic development, and binding of partition proteins at these sites regulates these processes. The family of N15-like linear phage-plasmids includes lambdoid phages ɸKO2 and pY54, as well as Myoviridae phages ΦHAP-1, VHML, VP882, Vp58.5, and vB_VpaM_MAR of marine gamma-proteobacteria. The genomes of these phages contain similar protelomerase genes, lysogeny control modules, and replication genes, suggesting that these phages may belong to a group diverged from a common ancestor.

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

    PubMed Central

    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

    2015-01-01

    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

  19. 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

  20. Phage fitness may help predict phage therapy efficacy

    PubMed Central

    Lindberg, Heather M; McKean, Kurt A; Wang, Ing-Nang

    2014-01-01

    We isolated 6 phages from 2 environmental water sources and assessed their ability to treat Pseudomonas aeruginosa infection of Drosophila melanogaster. We found all 6 phages were able to significantly increase mean survival time (MST) of infected D. melanogaster. Although phage traits, such as adsorption rate, burst size, and lysis time, varied significantly among these phages, none of the traits correlated significantly with MST. Phage growth rate determined in vitro, however, was found to be significantly correlated with MST. Overall, our study shows that infected D. melanogaster can be used as a model system to test the therapeutic efficacy of phages. In addition, a more comprehensive characteristic, like the in vitro growth rate, seems to be a better indicator in predicting therapeutic success than constituent traits like the adsorption rate, burst size, or lysis time. PMID:26713221

  1. Vibrio vulnificus Phage PV94 Is Closely Related to Temperate Phages of V. cholerae and Other Vibrio Species

    PubMed Central

    Reetz, Jochen; Strauch, Eckhard; Hertwig, Stefan

    2014-01-01

    Background Vibrio vulnificus is an important pathogen which can cause serious infections in humans. Yet, there is limited knowledge on its virulence factors and the question whether temperate phages might be involved in pathogenicity, as is the case with V. cholerae. Thus far, only two phages (SSP002 and VvAW1) infecting V. vulnificus have been genetically characterized. These phages were isolated from the environment and are not related to Vibrio cholerae phages. The lack of information on temperate V. vulnificus phages prompted us to isolate those phages from lysogenic strains and to compare them with phages of other Vibrio species. Results In this study the temperate phage PV94 was isolated from a V. vulnificus biotype 1 strain by mitomycin C induction. PV94 is a myovirus whose genome is a linear double-stranded DNA of 33,828 bp with 5′-protruding ends. Sequence analysis of PV94 revealed a modular organization of the genome. The left half of the genome comprising the immunity region and genes for the integrase, terminase and replication proteins shows similarites to V. cholerae kappa phages whereas the right half containing genes for structural proteins is closely related to a prophage residing in V. furnissii NCTC 11218. Conclusion We present the first genomic sequence of a temperate phage isolated from a human V. vulnificus isolate. The sequence analysis of the PV94 genome demonstrates the wide distribution of closely related prophages in various Vibrio species. Moreover, the mosaicism of the PV94 genome indicates a high degree of horizontal genetic exchange within the genus Vibrio, by which V. vulnificus might acquire virulence-associated genes from other species. PMID:24732980

  2. PLACENTAL GROWTH FACTOR ATTENUATES SUPPRESSION OF ERYTHROID COLONY FORMATION BY INTERFERON

    PubMed Central

    Dallalio, Gail; Means, Robert T.

    2008-01-01

    Placental growth factor (PlGF) is a member of the vascular endothelial growth factor family and is associated with inflammation and with pathologic angiogenesis. PlGF is released from marrow erythroid cells and serum PlGF concentrations have been reported to distinguish sickle cell patients from healthy controls. We observed that CFU-E from homozygous sickle cell (SS) patients are less sensitive to inhibition by recombinant human (rh) γ interferon (IFN) than those from healthy controls, and the contribution of PlGF to this process was evaluated. At concentrations 10 – 1000 pg/mL, PlGF neither inhibits nor enhances CFU-E colony formation, and there were no differences between the responses of SS patients or healthy controls. rhPlGF 100 pg/mL reversed the inhibitory effects of rhγIFN on CFU-E colony formation. rhPlGF significantly attenuated rhγIFN induction of Fas ligandin an erythroid cell line (HCD57). Both HCD57 cells and CD36+ human marrow cells express Flt-1, a receptor for PlGF. Neutralizing antibody against Flt-1 partially attenuated the IFN-protective effect of rhPlGF, although this effect was not statistically significant. In conclusion, increased PlGF concentrations in the marrow of SS patients may protect erythroid progenitors from cytokine-induced inhibition of colony formation, and may be a mechanism by which erythropoiesis in sickle cell disease is preserved despite concurrent inflammation. PMID:19010294

  3. 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

  4. Generalized transduction Of shigella flexneri by converting phage PE5.

    PubMed

    Financsek, I; Kétyi, I

    1976-01-01

    Phage PE5, responsible for the conversion of type V antigen in Shigella flexneri, has the ability to produce generalized transduction. The correlation between phage multiplicity and the number of transductants, the specific inhibitory activity of anti-PE5 serum, and the lack of transduction in PE5 resistant recipients, indicate the role of phage PE5 in generalized transduction. Transduction of the R100-1 factor resulted in a non-transmissible tetracycline resistance segragation. The characteristics of the tetracycline resistance determinant suggest the possibility of integration.

  5. Drugs derived from phage display

    PubMed Central

    Nixon, Andrew E; Sexton, Daniel J; Ladner, Robert C

    2014-01-01

    Phage display, one of today’s fundamental drug discovery technologies, allows identification of a broad range of biological drugs, including peptides, antibodies and other proteins, with the ability to tailor critical characteristics such as potency, specificity and cross-species binding. Further, unlike in vivo technologies, generating phage display-derived antibodies is not restricted by immunological tolerance. Although more than 20 phage display-derived antibody and peptides are currently in late-stage clinical trials or approved, there is little literature addressing the specific challenges and successes in the clinical development of phage-derived drugs. This review uses case studies, from candidate identification through clinical development, to illustrate the utility of phage display as a drug discovery tool, and offers a perspective for future developments of phage display technology. PMID:24262785

  6. Basic fibroblast growth factor attenuates the degeneration of injured spinal cord motor endplates

    PubMed Central

    Wang, Jianlong; Sun, Jianfeng; Tang, Yongxiang; Guo, Gangwen; Zhou, Xiaozhe; Chen, Yanliang; Shen, Minren

    2013-01-01

    The distal end of the spinal cord and neuromuscular junction may develop secondary degeneration and damage following spinal cord injury because of the loss of neural connections. In this study, a rat model of spinal cord injury, established using a modified Allen's method, was injected with basic fibroblast growth factor solution via subarachnoid catheter. After injection, rats with spinal cord injury displayed higher scores on the Basso, Beattie and Bresnahan locomotor scale. Motor function was also well recovered and hematoxylin-eosin staining showed that spinal glial scar hyperplasia was not apparent. Additionally, anterior tibial muscle fibers slowly, but progressively, atrophied. nohistochemical staining showed that the absorbance values of calcitonin gene related peptide and acetylcholinesterase in anterior tibial muscle and spinal cord were similar, and injection of basic broblast growth factor increased this absorbance. Results showed that after spinal cord injury, the distal motor neurons and motor endplate degenerated. Changes in calcitonin gene related peptide and acetylcholinesterase in the spinal cord anterior horn motor neurons and motor endplate then occurred that were consistent with this regeneration. Our findings indicate that basic fibroblast growth factor can protect the endplate through attenuating the decreased expression of calcitonin gene related peptide and acetylcholinesterase in anterior horn motor neurons of the injured spinal cord. PMID:25206531

  7. Fibroblast growth factor-1 attenuates TGF-β1-induced lung fibrosis.

    PubMed

    Shimbori, Chiko; Bellaye, Pierre-Simon; Xia, Jiaji; Gauldie, Jack; Ask, Kjetil; Ramos, Carlos; Becerril, Carina; Pardo, Annie; Selman, Moises; Kolb, Martin

    2016-10-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by progressive fibroblast and myofibroblast proliferation, and extensive deposition of extracellular matrix (ECM). Fibroblast growth factor-1 (FGF-1) belongs to the FGF family and has been shown to inhibit fibroblast collagen production and differentiation into myofibroblasts, and revert epithelial-mesenchymal transition by inhibiting TGF-β1 signalling pathways. However, the precise role of FGF-1 in pulmonary fibrosis has not yet been elucidated. In this study, we explore the mechanisms underlying the anti-fibrogenic effect of FGF-1 in pulmonary fibrosis in vitro and in vivo by prolonged transient overexpression of FGF-1 (AdFGF-1) and TGF-β1 (AdTGF-β1) using adenoviral vectors. In vivo, FGF-1 overexpression markedly attenuated TGF-β1-induced pulmonary fibrosis in rat lungs when given both concomitantly, or delayed, by enhancing proliferation and hyperplasia of alveolar epithelial cells (AECs). AdFGF-1 also attenuated the TGF-β1 signalling pathway and induced FGFR1 expression in AECs. In vitro, AdFGF-1 prevented the increase in α-SMA and the decrease in E-cadherin induced by AdTGF-β1 in normal human lung fibroblasts, primary human pulmonary AECs, and A549 cells. Concomitantly, AdTGF-β1-induced Smad2 phosphorylation was significantly reduced by AdFGF-1 in both cell types. AdFGF-1 also attenuated the increase in TGFβR1 protein and mRNA levels in fibroblasts. In AECs, AdFGF-1 decreased TGFβR1 protein by favouring TGFβR1 degradation through the caveolin-1/proteasome pathway. Furthermore, FGFR1 expression was increased in AECs, whereas it was decreased in fibroblasts. In serum of IPF patients, FGF-1 levels were increased compared to controls. Interestingly, FGF-1 expression was restricted to areas of AEC hyperplasia, but not α-SMA-positive areas in IPF lung tissue. Our results demonstrate that FGF-1 may have preventative and therapeutic effects on TGF-β1-driven pulmonary fibrosis via inhibiting

  8. Fibroblast growth factor-1 attenuates TGF-β1-induced lung fibrosis.

    PubMed

    Shimbori, Chiko; Bellaye, Pierre-Simon; Xia, Jiaji; Gauldie, Jack; Ask, Kjetil; Ramos, Carlos; Becerril, Carina; Pardo, Annie; Selman, Moises; Kolb, Martin

    2016-10-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by progressive fibroblast and myofibroblast proliferation, and extensive deposition of extracellular matrix (ECM). Fibroblast growth factor-1 (FGF-1) belongs to the FGF family and has been shown to inhibit fibroblast collagen production and differentiation into myofibroblasts, and revert epithelial-mesenchymal transition by inhibiting TGF-β1 signalling pathways. However, the precise role of FGF-1 in pulmonary fibrosis has not yet been elucidated. In this study, we explore the mechanisms underlying the anti-fibrogenic effect of FGF-1 in pulmonary fibrosis in vitro and in vivo by prolonged transient overexpression of FGF-1 (AdFGF-1) and TGF-β1 (AdTGF-β1) using adenoviral vectors. In vivo, FGF-1 overexpression markedly attenuated TGF-β1-induced pulmonary fibrosis in rat lungs when given both concomitantly, or delayed, by enhancing proliferation and hyperplasia of alveolar epithelial cells (AECs). AdFGF-1 also attenuated the TGF-β1 signalling pathway and induced FGFR1 expression in AECs. In vitro, AdFGF-1 prevented the increase in α-SMA and the decrease in E-cadherin induced by AdTGF-β1 in normal human lung fibroblasts, primary human pulmonary AECs, and A549 cells. Concomitantly, AdTGF-β1-induced Smad2 phosphorylation was significantly reduced by AdFGF-1 in both cell types. AdFGF-1 also attenuated the increase in TGFβR1 protein and mRNA levels in fibroblasts. In AECs, AdFGF-1 decreased TGFβR1 protein by favouring TGFβR1 degradation through the caveolin-1/proteasome pathway. Furthermore, FGFR1 expression was increased in AECs, whereas it was decreased in fibroblasts. In serum of IPF patients, FGF-1 levels were increased compared to controls. Interestingly, FGF-1 expression was restricted to areas of AEC hyperplasia, but not α-SMA-positive areas in IPF lung tissue. Our results demonstrate that FGF-1 may have preventative and therapeutic effects on TGF-β1-driven pulmonary fibrosis via inhibiting

  9. 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

  10. 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

  11. 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

  12. 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...

  13. Studies on a Smooth Phage-resistant Variant of Brucella abortus

    PubMed Central

    Corbel, M. J.; Morris, J. A.

    1974-01-01

    The immunological properties of a phage-resistant variant of Brucella abortus were examined. This variant, which was smooth and identical with the phage-susceptible parent strain in all cultural, biochemical and serological properties studied, was strongly antigenic in rabbits and guineapigs, inducing high titres of antibodies and delayed hypersensitivity to Br. abortus antigens. It was also fully virulent for mice and guinea-pigs. Contrary to other reports on the properties of phage-resistant Brucella strains, the in vivo properties of this strain showed no evidence of attenuation of virulence. PMID:4209104

  14. 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

  15. Ciprofloxacin and Trimethoprim Cause Phage Induction and Virulence Modulation in Staphylococcus aureus

    PubMed Central

    Goerke, Christiane; Köller, Johanna; Wolz, Christiane

    2006-01-01

    In Staphylococcus aureus strains of human origin, phages which integrate into the chromosomal gene coding for β-hemolysin (hlb) are widely distributed. Most of them encode accessory virulence determinants such as staphylokinase (sak) or enterotoxins. Here, we analyzed the effects of ciprofloxacin and trimethoprim on phage induction and expression of phage-encoded virulence factors by using isolates from patients with cystic fibrosis for which the induction of hlb-converting phages was demonstrated in vivo (C. Goerke, S. Matias y Papenberg, S. Dasbach, K. Dietz, R. Ziebach, B. C. Kahl, and C. Wolz, J. Infect. Dis. 189:724-734, 2004) as well as a φ13 lysogen of phage-cured strain 8325-4. Treatment of lysogens with subinhibitory concentrations of either antibiotic resulted in (i) delysogenization of strains resembling the isolates picked up after chronic lung infection and (ii) replication of phages in the bacterial host in a dose-dependent manner. Ciprofloxacin treatment resulted in enhanced recA transcription, indicating involvement of the SOS response in phage mobilization. Induction of φ13 was linked to elevated expression of the phage-encoded virulence gene sak, chiefly due to the activation of latent phage promoters. In summary, we could show the induction of hlb-converting phages and a subsequent virulence modulation of the host bacterium by ciprofloxacin and trimethoprim. PMID:16377683

  16. 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.

  17. 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

  18. Genomics of staphylococcal Twort-like phages--potential therapeutics of the post-antibiotic era.

    PubMed

    Łobocka, Małgorzata; Hejnowicz, Monika S; Dąbrowski, Kamil; Gozdek, Agnieszka; Kosakowski, Jarosław; Witkowska, Magdalena; Ulatowska, Magdalena I; Weber-Dąbrowska, Beata; Kwiatek, Magdalena; Parasion, Sylwia; Gawor, Jan; Kosowska, Helena; Głowacka, Aleksandra

    2012-01-01

    . Although the number and location of introns may vary between particular phages, intron shuffling is unlikely to be a major factor responsible for specificity differences.

  19. High stability of Stx2 phage in food and under food-processing conditions.

    PubMed

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

    2011-08-01

    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.

  20. Shiga toxins and stx phages: highly diverse entities.

    PubMed

    Krüger, Alejandra; Lucchesi, Paula M A

    2015-03-01

    Shiga toxins are the main virulence factors of a group of Escherichia coli strains [Shiga toxin-producing E. coli (STEC)] that cause severe human diseases, such as haemorrhagic colitis and haemolytic-uraemic syndrome. The Shiga toxin family comprises several toxin subtypes, which have been differentially related to clinical manifestations. In addition, the phages that carry the Shiga toxin genes (stx phages) are also diverse. These phages play an important role not only in the dissemination of Shiga toxin genes and the emergence of new STEC strains, but also in the regulation of Shiga toxin production. Consequently, differences in stx phages may affect the dissemination of stx genes as well as the virulence of STEC strains. In addition to presenting an overview of Shiga toxins and stx phages, in this review we highlight current knowledge about the diversity of stx phages, with emphasis on its impact on STEC virulence. We consider that this diversity should be taken into account when developing STEC infection treatments and diagnostic approaches, and when conducting STEC control in reservoirs. PMID:25479836

  1. Shiga toxins and stx phages: highly diverse entities.

    PubMed

    Krüger, Alejandra; Lucchesi, Paula M A

    2015-03-01

    Shiga toxins are the main virulence factors of a group of Escherichia coli strains [Shiga toxin-producing E. coli (STEC)] that cause severe human diseases, such as haemorrhagic colitis and haemolytic-uraemic syndrome. The Shiga toxin family comprises several toxin subtypes, which have been differentially related to clinical manifestations. In addition, the phages that carry the Shiga toxin genes (stx phages) are also diverse. These phages play an important role not only in the dissemination of Shiga toxin genes and the emergence of new STEC strains, but also in the regulation of Shiga toxin production. Consequently, differences in stx phages may affect the dissemination of stx genes as well as the virulence of STEC strains. In addition to presenting an overview of Shiga toxins and stx phages, in this review we highlight current knowledge about the diversity of stx phages, with emphasis on its impact on STEC virulence. We consider that this diversity should be taken into account when developing STEC infection treatments and diagnostic approaches, and when conducting STEC control in reservoirs.

  2. 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.

  3. 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

  4. Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections.

    PubMed

    Regeimbal, James M; Jacobs, Anna C; Corey, Brendan W; Henry, Matthew S; Thompson, Mitchell G; Pavlicek, Rebecca L; Quinones, Javier; Hannah, Ryan M; Ghebremedhin, Meron; Crane, Nicole J; Zurawski, Daniel V; Teneza-Mora, Nimfa C; Biswas, Biswajit; Hall, Eric R

    2016-10-01

    Multidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infection-associated morbidity. Interestingly, this effective cocktail is composed of four phages that do not kill the parent strain of the infection and one phage that simply delays bacterial growth in vitro via a strong but incomplete selection event. The cocktail here appears to function in a combinatorial manner, as one constituent phage targets capsulated A. baumannii bacteria and selects for loss of receptor, shifting the population to an uncapsulated state that is then sensitized to the remaining four phages in the cocktail. Additionally, capsule is a known virulence factor for A. baumannii, and we demonstrated that the emergent uncapsulated bacteria are avirulent in a Galleria mellonella model. These results highlight the importance of anticipating population changes during phage therapy and designing intelligent cocktails to control emergent strains, as well as the benefits of using phages that target virulence factors. Because of the efficacy of this cocktail isolated from a limited environmental pool, we have established a pipeline for developing new phage therapeutics against additional clinically relevant multidrug-resistant pathogens by using environmental phages sourced from around the globe.

  5. 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.

  6. 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

  7. The present position in Brucella phage research

    PubMed Central

    Droževkina, M. S.

    1963-01-01

    It is only comparatively recently that efforts to isolate and purify stable Brucella phages have met with success. This success has important implications for the epidemiology and possibly the control of brucellosis, and the author has therefore thought it opportune to summarize and review the present state of research on Brucella phages. After giving an outline of the history of Brucella phage isolation, she goes into the methodology of phage isolation from numerous sources, of phage reinforcement, and of determination of the lytic activity of phage. This is followed by a discussion of the biological properties, morphology and typing of Brucella phage, and of lysogeny in Brucella. Finally, she reviews the promising research that is being done on Brucella typing with specific phage and on the use of antiphage serum in diagnosis, and discusses the possible use of phage in the treatment and prevention of brucellosis. PMID:14043752

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. Phage Specificity of the Freshwater Fish Pathogen Flavobacterium columnare▿

    PubMed Central

    Laanto, Elina; Sundberg, Lotta-Riina; Bamford, Jaana K. H.

    2011-01-01

    Flavobacteria and their phages were isolated from Finnish freshwaters and fish farms. Emphasis was placed on finding phages infecting the fish pathogen Flavobacterium columnare for use as phage therapy agents. The host ranges of the flavobacterial phages varied, phages infecting F. columnare being more host specific than the other phages. PMID:21890667

  17. 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

  18. Complete Genome Sequence of Vibrio anguillarum Phage CHOED Successfully Used for Phage Therapy in Aquaculture

    PubMed Central

    Higuera, Gastón; Gajardo, Felipe; Castillo, Daniel; Middleboe, Mathias; García, Katherine; Ramírez, Carolina; Espejo, Romilio T.

    2014-01-01

    Vibrio anguillarum phage CHOED was isolated from Chilean mussels. It is a virulent phage showing effective inhibition of V. anguillarum. CHOED has potential in phage therapy, because it can protect fish from vibriosis in fish farms. Here, we announce the completely sequenced genome of V. anguillarum phage CHOED. PMID:25013148

  19. Phage Community Dynamics in Hot Springs

    PubMed Central

    Breitbart, Mya; Wegley, Linda; Leeds, Steven; Schoenfeld, Tom; Rohwer, Forest

    2004-01-01

    In extreme thermal environments such as hot springs, phages are the only known microbial predators. Here we present the first study of prokaryotic and phage community dynamics in these environments. Phages were abundant in hot springs, reaching concentrations of a million viruses per milliliter. Hot spring phage particles were resistant to shifts to lower temperatures, possibly facilitating DNA transfer out of these extreme environments. The phages were actively produced, with a population turnover time of 1 to 2 days. Phage-mediated microbial mortality was significant, making phage lysis an important component of hot spring microbial food webs. Together, these results show that phages exert an important influence on microbial community structure and energy flow in extreme thermal environments. PMID:15006788

  20. 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

  1. Myocardial Connective Tissue Growth Factor (CCN2/CTGF) Attenuates Left Ventricular Remodeling after Myocardial Infarction

    PubMed Central

    Gravning, Jørgen; Ørn, Stein; Kaasbøll, Ole Jørgen; Martinov, Vladimir N.; Manhenke, Cord; Dickstein, Kenneth; Edvardsen, Thor; Attramadal, Håvard; Ahmed, Mohammed Shakil

    2012-01-01

    Aims Myocardial CCN2/CTGF is induced in heart failure of various etiologies. However, its role in the pathophysiology of left ventricular (LV) remodeling after myocardial infarction (MI) remains unresolved. The current study explores the role of CTGF in infarct healing and LV remodeling in an animal model and in patients admitted for acute ST-elevation MI. Methods and Results Transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) and non-transgenic littermate controls (NLC) were subjected to permanent ligation of the left anterior descending coronary artery. Despite similar infarct size (area of infarction relative to area at risk) 24 hours after ligation of the coronary artery in Tg-CTGF and NLC mice, Tg-CTGF mice disclosed smaller area of scar tissue, smaller increase of cardiac hypertrophy, and less LV dilatation and deterioration of LV function 4 weeks after MI. Tg-CTGF mice also revealed substantially reduced mortality after MI. Remote/peri-infarct tissue of Tg-CTGF mice contained reduced numbers of leucocytes, macrophages, and cells undergoing apoptosis as compared with NLC mice. In a cohort of patients with acute ST-elevation MI (n = 42) admitted to hospital for percutaneous coronary intervention (PCI) serum-CTGF levels (s-CTGF) were monitored and related to infarct size and LV function assessed by cardiac MRI. Increase in s-CTGF levels after MI was associated with reduced infarct size and improved LV ejection fraction one year after MI, as well as attenuated levels of CRP and GDF-15. Conclusion Increased myocardial CTGF activities after MI are associated with attenuation of LV remodeling and improved LV function mediated by attenuation of inflammatory responses and inhibition of apoptosis. PMID:23284892

  2. 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

  3. Phage therapy of the white plague-like disease of Favia favus in the Red Sea

    NASA Astrophysics Data System (ADS)

    Atad, I.; Zvuloni, A.; Loya, Y.; Rosenberg, E.

    2012-09-01

    Coral disease is a major factor in the global decline of coral reefs. At present, there are no known procedures for preventing or treating infectious diseases of corals. Immunization is not possible because corals have a restricted adaptive immune system and antibiotics are neither ecologically safe nor practical in an open system. Thus, we tested phage therapy as an alternative therapeutic method for treating diseased corals. Phage BA3, specific to the coral pathogen Thalassomonas loyana, inhibited the progression of the white plague-like disease and transmission to healthy corals in the Gulf of Aqaba, Red Sea. Only one out of 19 (5 %) of the healthy corals became infected when placed near phage-treated diseased corals, whereas 11 out of 18 (61 %) healthy corals were infected in the no-phage control. This is the first successful treatment for a coral disease in the sea. We posit that phage therapy of certain coral diseases is achievable in situ.

  4. Controllable attenuators

    NASA Astrophysics Data System (ADS)

    Krylov, G. M.; Khoniak, E. I.; Tynynyka, A. N.; Iliushenko, V. N.; Sikolenko, S. F.

    Methods for the synthesis of controllable attenuators and their implementations are examined. In particular, attention is given to the general properties of controllable attenuators, control elements, types of controllable attenuators and methods of their analysis, and synthesis of the control characteristic of attenuators. The discussion also covers the efficiency of attenuator control, the use of transmission line segments in wide-band controllable attenuators, and attenuators with a discretely controlled transmission coefficient.

  5. Revenge of the phages: defeating bacterial defences.

    PubMed

    Samson, Julie E; Magadán, Alfonso H; Sabri, Mourad; Moineau, Sylvain

    2013-10-01

    Bacteria and their viral predators (bacteriophages) are locked in a constant battle. In order to proliferate in phage-rich environments, bacteria have an impressive arsenal of defence mechanisms, and in response, phages have evolved counter-strategies to evade these antiviral systems. In this Review, we describe the various tactics that are used by phages to overcome bacterial resistance mechanisms, including adsorption inhibition, restriction-modification, CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems and abortive infection. Furthermore, we consider how these observations have enhanced our knowledge of phage biology, evolution and phage-host interactions. PMID:23979432

  6. N-Terminal Labeling Of Filamentous Phage To Create Cancer Marker Imaging Agents

    PubMed Central

    Carrico, Zachary M.; Farkas, Michelle E.; Zhou, Yu; Hsiao, Sonny C.; Marks, James D.; Chokhawala, Harshal; Clark, Douglas S.; Francis, Matthew B.

    2012-01-01

    We report a convenient new technique for the labeling of filamentous phage capsid proteins. Previous reports have shown that phage coat protein residues can be modified, but the lack of chemically distinct amino acids in the coat protein sequences makes it difficult to attach high levels of synthetic molecules without altering the binding capabilities of the phage. To modify the phage with polymer chains, imaging groups, and other molecules, we have developed chemistry to convert the N-terminal amines of the ~4,200 coat proteins into ketone groups. These sites can then serve as chemospecific handles for the attachment of alkoxyamine groups through oxime formation. Specifically, we demonstrate the attachment of fluorophores and up to 3,000 molecules of 2 kD poly(ethylene glycol) (PEG2k) to each of the phage capsids without significantly affecting the binding of phage-displayed antibody fragments to EGFR and HER2 (two important epidermal growth factor receptors). We also demonstrate the utility of the modified phage for the characterization of breast cancer cells using multicolor fluorescence microscopy. Due to the widespread use of filamentous phage as display platforms for peptide and protein evolution, we envision that the ability to attach large numbers of synthetic functional groups to their coat proteins will be of significant value to the biological and materials communities. PMID:22830952

  7. Comparative Persistence of Subgroups of F-Specific RNA Phages in River Water

    PubMed Central

    Yang, Yongheng

    2013-01-01

    F-specific (F+) RNA phages are widely used as indicators for the presence of fecal contamination and/or enteric viruses in water, and identifying subgroups of F+ RNA phages provides an approach for microbial source tracking. Different survival characteristics of the F+ RNA phage subgroups result in a misinterpretation of their original proportion in water, thus giving misleading information when they are used for microbial source tracking. This study investigated the comparative persistence of subgroups of F+ RNA phages in river water under different conditions. Results suggested that temperature and pH are the major factors affecting the persistence of F+ RNA phages in river water, and organic substances promote phage survival. The comparative persistence patterns of subgroups of F+ RNA phages varied and may bias extrapolation of their initial proportions in surface water. Thus, the characteristics of water should be taken into consideration and the results should be carefully interpreted when F+ RNA phages are used for microbial source tracking. PMID:23686274

  8. Phage typing of Staphylococcus saprophyticus.

    PubMed Central

    Torres Pereira, A.; Melo Cristino, J. A.

    1991-01-01

    This study included 502 staphylococcus strains; Staphylococcus saprophyticus (297 strains) S. cohnii (47), S. xylosus (10), S. epidermidis (67) and S. aureus (81). Mitomycin C induction was performed on 100 isolates of S. saprophyticus and all induced strains were reacted with each other. Twenty-six strains proved to be lysogenic. Phages were propagated and titrated. With 12 of the phages there were three frequent associations, named lytic groups A, B and C, which included 75% of all typable strains. Typability of the system was 45% and reproducibility was between 94.2% and 100%. Phages did not lyse S. aureus and S. epidermidis strains, but they lysed S. saprophyticus and only rare strains of other novobiocin resistant species. Effective S. saprophyticus typing serves ecological purposes and tracing the origin of urinary strains from the skin or mucous membranes. Phage typing in association with plasmid profiling previously described, are anticipated as complementary methods with strong discriminatory power for differentiating among S. saprophyticus strains. PMID:1752305

  9. 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

  10. Streptococcus pyogenes CAMP factor attenuates phagocytic activity of RAW 264.7 cells.

    PubMed

    Kurosawa, Mie; Oda, Masataka; Domon, Hisanori; Saitoh, Issei; Hayasaki, Haruaki; Terao, Yutaka

    2016-02-01

    Streptococcus pyogenes produces molecules that inhibit the function of human immune system, thus allowing the pathogen to grow and spread in tissues. It is known that S. pyogenes CAMP factor increases erythrocytosis induced by Staphylococcus aureus β-hemolysin. However, the effects of CAMP factor for immune cells are unclear. In this study, we investigated the effects of CAMP factor to macrophages. Western blotting analysis demonstrated that all examined strains expressed CAMP factor protein. In the presence of calcium or magnesium ion, CAMP factor was significantly released in the supernatant. In addition, both culture supernatant from S. pyogenes strain SSI-9 and recombinant CAMP factor dose-dependently induced vacuolation in RAW 264.7 cells, but the culture supernatant from Δcfa isogenic mutant strain did not. CAMP factor formed oligomers in RAW 264.7 cells in a time-dependent manner. CAMP factor suppressed cell proliferation via G2 phase cell cycle arrest without inducing cell death. Furthermore, CAMP factor reduced the uptake of S. pyogenes and phagocytic activity indicator by RAW 264.7 cells. These results suggest that CAMP factor works as a macrophage dysfunction factor. Therefore, we conclude that CAMP factor allows S. pyogenes to escape the host immune system, and contribute to the spread of streptococcal infection.

  11. The habits of highly effective phages: population dynamics as a framework for identifying therapeutic phages.

    PubMed

    Bull, James J; Gill, Jason J

    2014-01-01

    The use of bacteriophages as antibacterial agents is being actively researched on a global scale. Typically, the phages used are isolated from the wild by plating on the bacteria of interest, and a far larger set of candidate phages is often available than can be used in any application. When an excess of phages is available, how should the best phages be identified? Here we consider phage-bacterial population dynamics as a basis for evaluating and predicting phage success. A central question is whether the innate dynamical properties of phages are the determinants of success, or instead, whether extrinsic, indirect effects can be responsible. We address the dynamical perspective, motivated in part by the absence of dynamics in previously suggested principles of phage therapy. Current mathematical models of bacterial-phage dynamics do not capture the realities of in vivo dynamics, nor is this likely to change, but they do give insight to qualitative properties that may be generalizable. In particular, phage adsorption rate may be critical to treatment success, so understanding the effects of the in vivo environment on host availability may allow prediction of useful phages prior to in vivo experimentation. Principles for predicting efficacy may be derived by developing a greater understanding of the in vivo system, or such principles could be determined empirically by comparing phages with known differences in their dynamic properties. The comparative approach promises to be a powerful method of discovering the key to phage success. We offer five recommendations for future study: (i) compare phages differing in treatment efficacy to identify the phage properties associated with success, (ii) assay dynamics in vivo, (iii) understand mechanisms of bacterial escape from phages, (iv) test phages in model infections that are relevant to the intended clinical applications, and (v) develop new classes of models for phage growth in spatially heterogeneous environments

  12. Systematic annotation and analysis of "virmugens"-virulence factors whose mutants can be used as live attenuated vaccines.

    PubMed

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

    2013-01-21

    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 1 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, 6 are related to carbohydrate or nucleotide transport and metabolism, and 2 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 to elucidate enriched virmugen profiles and the mechanisms of protective immunity, and further supports rational vaccine design.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

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

    PubMed

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

    2016-06-16

    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.

  2. 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.

  3. 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

  4. Phage-encoded Serine Integrases and Other Large Serine Recombinases.

    PubMed

    Smith, Margaret C M

    2015-08-01

    The large serine recombinases (LSRs) are a family of enzymes, encoded in temperate phage genomes or on mobile elements, that precisely cut and recombine DNA in a highly controllable and predictable way. In phage integration, the LSRs act at specific sites, the attP site in the phage and the attB site in the host chromosome, where cleavage and strand exchange leads to the integrated prophage flanked by the recombinant sites attL and attR. The prophage can excise by recombination between attL and attR but this requires a phage-encoded accessory protein, the recombination directionality factor (RDF). Although the LSRs can bind specifically to all the recombination sites, only specific integrase-bound sites can pair in a synaptic complex prior to strand exchange. Recent structural information has led to a breakthrough in our understanding of the mechanism of the LSRs, notably how the LSRs bind to their substrates and how LSRs display this site-selectivity. We also understand that the RDFs exercise control over the LSRs by protein-protein interactions. Other recent work with the LSRs have contributed to our understanding of how all serine recombinases undergo strand exchange subunit rotation, facilitated by surfaces that resemble a molecular bearing.

  5. 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

  6. 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

  7. Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections

    PubMed Central

    Regeimbal, James M.; Jacobs, Anna C.; Corey, Brendan W.; Henry, Matthew S.; Thompson, Mitchell G.; Pavlicek, Rebecca L.; Quinones, Javier; Hannah, Ryan M.; Ghebremedhin, Meron; Crane, Nicole J.; Zurawski, Daniel V.; Teneza-Mora, Nimfa C.; Hall, Eric R.

    2016-01-01

    Multidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infection-associated morbidity. Interestingly, this effective cocktail is composed of four phages that do not kill the parent strain of the infection and one phage that simply delays bacterial growth in vitro via a strong but incomplete selection event. The cocktail here appears to function in a combinatorial manner, as one constituent phage targets capsulated A. baumannii bacteria and selects for loss of receptor, shifting the population to an uncapsulated state that is then sensitized to the remaining four phages in the cocktail. Additionally, capsule is a known virulence factor for A. baumannii, and we demonstrated that the emergent uncapsulated bacteria are avirulent in a Galleria mellonella model. These results highlight the importance of anticipating population changes during phage therapy and designing intelligent cocktails to control emergent strains, as well as the benefits of using phages that target virulence factors. Because of the efficacy of this cocktail isolated from a limited environmental pool, we have established a pipeline for developing new phage therapeutics against additional clinically relevant multidrug-resistant pathogens by using environmental phages sourced from around the globe. PMID:27431214

  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. Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections.

    PubMed

    Regeimbal, James M; Jacobs, Anna C; Corey, Brendan W; Henry, Matthew S; Thompson, Mitchell G; Pavlicek, Rebecca L; Quinones, Javier; Hannah, Ryan M; Ghebremedhin, Meron; Crane, Nicole J; Zurawski, Daniel V; Teneza-Mora, Nimfa C; Biswas, Biswajit; Hall, Eric R

    2016-10-01

    Multidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infection-associated morbidity. Interestingly, this effective cocktail is composed of four phages that do not kill the parent strain of the infection and one phage that simply delays bacterial growth in vitro via a strong but incomplete selection event. The cocktail here appears to function in a combinatorial manner, as one constituent phage targets capsulated A. baumannii bacteria and selects for loss of receptor, shifting the population to an uncapsulated state that is then sensitized to the remaining four phages in the cocktail. Additionally, capsule is a known virulence factor for A. baumannii, and we demonstrated that the emergent uncapsulated bacteria are avirulent in a Galleria mellonella model. These results highlight the importance of anticipating population changes during phage therapy and designing intelligent cocktails to control emergent strains, as well as the benefits of using phages that target virulence factors. Because of the efficacy of this cocktail isolated from a limited environmental pool, we have established a pipeline for developing new phage therapeutics against additional clinically relevant multidrug-resistant pathogens by using environmental phages sourced from around the globe. PMID:27431214

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. Experimental Phage Therapy for Burkholderia pseudomallei Infection.

    PubMed

    Guang-Han, Ong; 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

  16. Use of phages to control Campylobacter spp.

    PubMed

    Janež, Nika; Loc-Carrillo, Catherine

    2013-10-01

    The use of phages to control pathogenic bacteria has been investigated since they were first discovered in the beginning of the 1900s. Over the last century we have slowly gained an in-depth understanding of phage biology including which phage properties are desirable when considering phage as biocontrol agents and which phage characteristics to potentially avoid. Campylobacter infections are amongst the most frequently encountered foodborne bacterial infections around the world. Handling and consumption of raw or undercooked poultry products have been determined to be the main route of transmission. The ability to use phages to target these bacteria has been studied for more than a decade and although we have made progress towards deciphering how best to use phages to control Campylobacter associated with poultry production, there is still much work to be done. This review outlines methods to improve the isolation of these elusive phages, as well as methods to identify desirable characteristics needed for a successful outcome. It also highlights the body of research undertaken so far and what criteria to consider when doing in-vivo studies, especially because some in-vitro studies have not been found to translate into to phage efficacy in-vivo.

  17. Attenuation of fibroblast growth factor signaling by poly-N-acatyllactosamine type glycans

    PubMed Central

    Sugihara, Kazuhiro; Shibata, Toshiaki K.; Takata, Kayoko; Kimura, Takako; Kanayama, Naohiro; Williams, Roy; Hatakeyama, Shingo; Akama, Tomoya O.; Kuo, Chu-Wei; Khoo, Kay-Hooi; Fukuda, Michiko N.

    2014-01-01

    Fibroblast growth factors (FGFs) and their receptors are expressed in a variety of mammalian tissues, playing a role in development and cell proliferation. While analyzing human sperm motility, we found that sperm treated with endo-β-galactosidase (EBG), which specifically hydrolyzes poly-N-acetyllactosamine type glycans (polyLacs), enhanced motility. Mass spectrometry analysis revealed that sperm-associated polyLacs are heavily fucosylated, consistent with Lewis Y antigen. Immunohistochemistry of epididymis using an anti-Lewis Y antibody before and after EBG treatment suggested that polyLacs carrying the Lewis Y epitope are synthesized in epididymal epithelia and secreted to seminal fluid. EBG-treated sperm elevated cAMP levels and calcium influx, indicating activation of fibroblast growth factor signaling. Seminal fluid polyLacs bound to FGFs in vitro, and impaired FGF-mediated signaling in HEK293T cells. PMID:23968720

  18. Heparin-binding epidermal growth factor–like growth factor attenuates acute lung injury and multiorgan dysfunction after scald burn

    PubMed Central

    Lutmer, Jeffrey; Watkins, Daniel; Chen, Chun-Liang; Velten, Markus; Besner, Gail

    2013-01-01

    Background Impaired gut barrier function and acute lung injury (ALI) are significant components of the multiorgan dysfunction syndrome that accompanies severe burns. Heparin-binding epidermal growth factor–like growth factor (HB-EGF) has been shown to reduce inflammation, preserve gut barrier function, and protect the lungs from acute injury in several models of intestinal injury; however, comparable effects of HB-EGF after burn injury have never been investigated. The present studies were based on the hypothesis that HB-EGF would reduce the severity of ALI and multiorgan dysfunction after scald burns in mice. Materials and methods Mice were randomized to sham, burn (25% of total body surface area with full thickness dorsal scald), and burn + HB-EGF groups. The HB-EGF group was pre-treated with two enteral doses of HB-EGF (1200 μg/kg/dose). Mice were resuscitated after injury and sacrificed at 8 h later. Their lungs were harvested for determination of pulmonary myeloperoxidase activity, wet:dry ratios, and terminal deoxynucleotidyl transferase dUTP nick end label and cleaved caspase 3 immunohistochemistry. Lung function was assessed using the SCIREQ Flexivent. Splenic apoptosis was quantified by Western blot for cleaved caspase 3, and intestinal permeability was measured using the everted gut sac method. Results Mice subjected to scald burn injury had increased lung myeloperoxidase levels, increased pulmonary and splenic apoptosis, elevated airway resistance and bronchial reactivity, and increased intestinal permeability compared with sham mice. These abnormalities were significantly attenuated in mice that were subjected to scald burn injury but treated with enteral HB-EGF. Conclusions These data suggest that HB-EGF protects mice from ALI after scald burn and attenuates the severity of postburn multiorgan dysfunction. PMID:23777985

  19. 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

  20. 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

  1. 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

  2. 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.

  3. Contribution of phage-derived genomic islands to the virulence of facultative bacterial pathogens.

    PubMed

    Busby, Ben; Kristensen, David M; Koonin, Eugene V

    2013-02-01

    Facultative pathogens have extremely dynamic pan-genomes, to a large extent derived from bacteriophages and other mobile elements. We developed a simple approach to identify phage-derived genomic islands and apply it to show that pathogens from diverse bacterial genera are significantly enriched in clustered phage-derived genes compared with related benign strains. These findings show that genome expansion by integration of prophages containing virulence factors is a major route of evolution of facultative bacterial pathogens.

  4. 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.

  5. Orthopedic surgery and bone fracture pain are both significantly attenuated by sustained blockade of nerve growth factor.

    PubMed

    Majuta, Lisa A; Longo, Geraldine; Fealk, Michelle N; McCaffrey, Gwen; Mantyh, Patrick W

    2015-01-01

    The number of patients suffering from postoperative pain due to orthopedic surgery and bone fracture is projected to dramatically increase because the human life span, weight, and involvement in high-activity sports continue to rise worldwide. Joint replacement or bone fracture frequently results in skeletal pain that needs to be adequately controlled for the patient to fully participate in needed physical rehabilitation. Currently, the 2 major therapies used to control skeletal pain are nonsteroidal anti-inflammatory drugs and opiates, both of which have significant unwanted side effects. To assess the efficacy of novel therapies, mouse models of orthopedic and fracture pain were developed and evaluated here. These models, orthopedic surgery pain and bone fracture pain, resulted in skeletal pain-related behaviors that lasted 3 weeks and 8 to 10 weeks, respectively. These skeletal pain behaviors included spontaneous and palpation-induced nocifensive behaviors, dynamic weight bearing, limb use, and voluntary mechanical loading of the injured hind limb. Administration of anti-nerve growth factor before orthopedic surgery or after bone fracture attenuated skeletal pain behaviors by 40% to 70% depending on the end point being assessed. These data suggest that nerve growth factor is involved in driving pain due to orthopedic surgery or bone fracture. These animal models may be useful in developing an understanding of the mechanisms that drive postoperative orthopedic and bone fracture pain and the development of novel therapies to treat these skeletal pains.

  6. Orthopedic surgery and bone fracture pain are both significantly attenuated by sustained blockade of nerve growth factor

    PubMed Central

    Majuta, Lisa A.; Longo, Geraldine; Fealk, Michelle N.; McCaffrey, Gwen; Mantyh, Patrick W.

    2015-01-01

    The number of patients suffering from postoperative pain due to orthopedic surgery and bone fracture is projected to dramatically increase because the human life span, weight, and involvement in high-activity sports continue to rise worldwide. Joint replacement or bone fracture frequently results in skeletal pain that needs to be adequately controlled for the patient to fully participate in needed physical rehabilitation. Currently, the 2 major therapies used to control skeletal pain are nonsteroidal anti-inflammatory drugs and opiates, both of which have significant unwanted side effects. To assess the efficacy of novel therapies, mouse models of orthopedic and fracture pain were developed and evaluated here. These models, orthopedic surgery pain and bone fracture pain, resulted in skeletal pain–related behaviors that lasted 3 weeks and 8 to 10 weeks, respectively. These skeletal pain behaviors included spontaneous and palpation-induced nocifensive behaviors, dynamic weight bearing, limb use, and voluntary mechanical loading of the injured hind limb. Administration of anti–nerve growth factor before orthopedic surgery or after bone fracture attenuated skeletal pain behaviors by 40% to 70% depending on the end point being assessed. These data suggest that nerve growth factor is involved in driving pain due to orthopedic surgery or bone fracture. These animal models may be useful in developing an understanding of the mechanisms that drive postoperative orthopedic and bone fracture pain and the development of novel therapies to treat these skeletal pains. PMID:25599311

  7. Gonadotropin surge-inhibiting/attenuating factors: a review of current evidence, potential applications, and future directions for research.

    PubMed

    Vega, Mario G; Zarek, Shvetha M; Bhagwat, Medha; Segars, James H

    2015-01-01

    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.

  8. 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

  9. 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

  10. Environmental factors influencing attenuation of methane and hydrochlorofluorocarbons in landfill cover soils.

    PubMed

    Scheutz, Charlotte; Kjeldsen, Peter

    2004-01-01

    The influence of different environmental factors on methane oxidation and degradation of hydrochlorofluorocarbons (HCFCs) was investigated in microcosms containing soil sampled at Skellingsted Landfill, Denmark. The soil showed a high capacity for methane oxidation resulting in a maximum oxidation rate of 104 microg CH4 g(-1) h(-1) and a low affinity of methane with a half-saturation constant of 2.0% v/v. The hydrochlorofluorocarbons HCFC-21 (dichlorofluoromethane) and HCFC-22 (chlorodifluoromethane) were rapidly oxidized and the oxidation occurred in parallel with the oxidation of methane. The maximal HCFC oxidation rates were 0.95 and 0.68 microg g(-1) h(-1) for HCFC-21 and HCFC-22, respectively. Increasing concentrations of HCFCs resulted in decreased methane oxidation rates. However, compared with typical concentrations in landfill gas, relatively high HCFC concentrations were needed to obtain a significant inhibition of methane oxidation. In general, the environmental factors studied influenced the degradation of HCFCs in almost the same way as they influenced methane oxidation. Temperature had a strong influence on the methanotrophic activity giving high Q10 values of 3.4 to 4.1 over the temperature range of 2 to 25 degrees C. Temperature optimum was around 30 degrees C; however, oxidation occurred at temperatures as low as 2 degrees C. A moisture content of 25% w/w yielded the maximum oxidation rate as it allowed good gas transport together with sufficient microbial activity. The optimum pH was around neutrality (pH = 6.5-7.5) showing that the methanotrophs were optimally adapted to the in situ pH, which was 6.9. Copper showed no inhibitory effect when added in relatively high concentrations (up to 60 mg kg(-1)), most likely due to sorption of copper ions to soil particles. At higher copper concentrations the oxidation rates decreased. The oxidation rates for methane, HCFC-21, and HCFC-22 were unaltered in ammonium-amended soil up to 14 mg kg(-1). Higher

  11. Environmental factors influencing attenuation of methane and hydrochlorofluorocarbons in landfill cover soils.

    PubMed

    Scheutz, Charlotte; Kjeldsen, Peter

    2004-01-01

    The influence of different environmental factors on methane oxidation and degradation of hydrochlorofluorocarbons (HCFCs) was investigated in microcosms containing soil sampled at Skellingsted Landfill, Denmark. The soil showed a high capacity for methane oxidation resulting in a maximum oxidation rate of 104 microg CH4 g(-1) h(-1) and a low affinity of methane with a half-saturation constant of 2.0% v/v. The hydrochlorofluorocarbons HCFC-21 (dichlorofluoromethane) and HCFC-22 (chlorodifluoromethane) were rapidly oxidized and the oxidation occurred in parallel with the oxidation of methane. The maximal HCFC oxidation rates were 0.95 and 0.68 microg g(-1) h(-1) for HCFC-21 and HCFC-22, respectively. Increasing concentrations of HCFCs resulted in decreased methane oxidation rates. However, compared with typical concentrations in landfill gas, relatively high HCFC concentrations were needed to obtain a significant inhibition of methane oxidation. In general, the environmental factors studied influenced the degradation of HCFCs in almost the same way as they influenced methane oxidation. Temperature had a strong influence on the methanotrophic activity giving high Q10 values of 3.4 to 4.1 over the temperature range of 2 to 25 degrees C. Temperature optimum was around 30 degrees C; however, oxidation occurred at temperatures as low as 2 degrees C. A moisture content of 25% w/w yielded the maximum oxidation rate as it allowed good gas transport together with sufficient microbial activity. The optimum pH was around neutrality (pH = 6.5-7.5) showing that the methanotrophs were optimally adapted to the in situ pH, which was 6.9. Copper showed no inhibitory effect when added in relatively high concentrations (up to 60 mg kg(-1)), most likely due to sorption of copper ions to soil particles. At higher copper concentrations the oxidation rates decreased. The oxidation rates for methane, HCFC-21, and HCFC-22 were unaltered in ammonium-amended soil up to 14 mg kg(-1). Higher

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

    PubMed

    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

  13. 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

  14. 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.

  15. 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

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

    PubMed

    Malarkey, Christopher S; Gustafson, Claire E; Saifee, Jessica F; Torres, Raul M; Churchill, Mair E A; 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

  17. Granulocyte/macrophage colony-stimulating factor attenuates endothelial hyperpermeability after thermal injury.

    PubMed

    Zhao, Jingling; Chen, Lei; Shu, Bin; Tang, Jinming; Zhang, Lijun; Xie, Julin; Liu, Xusheng; Xu, Yingbin; Qi, Shaohai

    2015-01-01

    Microvascular hyperpermeability followed by burn injury is the main cause of shock, and cardiovascular collapse can result if the condition is treated improperly. Our previous studies demonstrated that granulocyte/macrophage colony-stimulating factor (GM-CSF) clearly reduces microvascular permeability and protects microvessels against burn injury. However, the mechanism underlying the protective function of GM-CSF on burn-injured microvessels remains unknown. This study aimed to investigate the effect and mechanism of GM-CSF on endothelial cells after exposure to burn serum. We demonstrated that GM-CSF reduced post-burn endothelial "capillary leak" by inhibiting the activity of RhoA and maintaining the membrane localization of VE-cadherin. Membranous VE-cadherin enhances adherens junctions between endothelial cells and co-localizes with and activates VEGFR2, which protect cells from burn serum-induced apoptosis. Our findings suggest that the protective mechanism of GM-CSF on burn serum-injured endothelial monolayer hyperpermeability is achieved by strengthening cell adherens junctions and improving cell viability.

  18. 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

  19. [Phage therapy for bacterial infection of burn].

    PubMed

    Peng, Y Z; Huang, G T

    2016-09-20

    With the long-term and widespread use of antibiotics, drug resistance of bacteria has become a major problem in the treatment of burn infection. For treating multidrug resistant bacteria, phage therapy has become the focus of attention. Development of phage therapy to fill the blank of this field in China is extremely urgent. PMID:27647065

  20. 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

  1. 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

  2. Human recombinant factor VIIa may improve heat intolerance in mice by attenuating hypothalamic neuronal apoptosis and damage.

    PubMed

    Hsu, Chuan-Chih; Chen, Sheng-Hsien; Lin, Cheng-Hsien; Yung, Ming-Chi

    2014-10-01

    Intolerance to heat exposure is believed to be associated with hypothalamo-pituitary-adrenocortical (HPA) axis impairment [reflected by decreases in blood concentrations of both adrenocorticotrophic-hormone (ACTH) and corticosterone]. The purpose of this study was to determine the effect of human recombinant factor VIIa (rfVIIa) on heat intolerance, HPA axis impairment, and hypothalamic inflammation, ischemic and oxidative damage, and apoptosis in mice under heat stress. Immediately after heat stress (41.2 °C for 1 h), mice were treated with vehicle (1 mL/kg of body weight) or rfVIIa (65-270 µg/kg of body weight) and then returned to room temperature (26 °C). Mice still alive on day 4 of heat exposure were considered survivors. Cellular ischemia markers (e.g., glutamate, lactate-to-pyruvate ratio), oxidative damage markers (e.g., nitric oxide metabolite, hydroxyl radials), and pro-inflammatory cytokines (e.g., interleukin-6, interleukin-1β, tumor necrosis factor-α) in hypothalamus were determined. In addition, blood concentrations of both ACTH and corticosterone were measured. Hypothalamic cell damage was assessed by determing the neuronal damage scores, whereas the hypothalamic cell apoptosis was determined by assessing the numbers of cells stained with terminal deoxynucleotidyl transferase-mediated αUTP nick-end labeling, caspase-3-positive cells, and platelet endothelial cell adhesion molecula-1-positive cells in hypothalamus. Compared with vehicle-treated heated mice, rfVIIa-treated heated mice had significantly higher fractional survival (8/10 vs 1/10), lesser thermoregulatory deficit (34.1 vs 24.8 °C), lesser extents of ischemic, oxidative, and inflammatory markers in hypothalamus, lesser neuronal damage scores and apoptosis in hypothalamus, and lesser HPA axis impairment. Human recombinant factor VIIa appears to exert a protective effect against heatstroke by attenuating hypothalamic cell apoptosis (due to ischemic, inflammatory, and oxidative damage

  3. 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

  4. Bacteriophage T4 Infection of Stationary Phase E. coli: Life after Log from a Phage Perspective

    PubMed Central

    Bryan, Daniel; El-Shibiny, Ayman; Hobbs, Zack; Porter, Jillian; Kutter, Elizabeth M.

    2016-01-01

    Virtually all studies of phage infections investigate bacteria growing exponentially in rich media. In nature, however, phages largely encounter non-growing cells. Bacteria entering stationary phase often activate well-studied stress defense mechanisms that drastically alter the cell, facilitating its long-term survival. An understanding of phage-host interactions in such conditions is of major importance from both an ecological and therapeutic standpoint. Here, we show that bacteriophage T4 can efficiently bind to, infect and kill E. coli in stationary phase, both in the presence and absence of a functional stationary-phase sigma factor, and explore the response of T4-infected stationary phase cells to the addition of fresh nutrients 5 or 24 h after that infection. An unexpected new mode of response has been identified. “Hibernation” mode is a persistent but reversible dormant state in which the infected cells make at least some phage enzymes, but halt phage development until appropriate nutrients become available before producing phage particles. Our evidence indicates that the block in hibernation mode occurs after the middle-mode stage of phage development; host DNA breakdown and the incorporation of the released nucleotides into phage DNA indicate that the enzymes of the nucleotide synthesizing complex, under middle-mode control, have been made and assembled into a functional state. Once fresh glucose and amino acids become available, the standard lytic infection process rapidly resumes and concentrations of up to 1011 progeny phage (an average of about 40 phage per initially present cell) are produced. All evidence is consistent with the hibernation-mode control point lying between middle mode and late mode T4 gene expression. We have also observed a “scavenger” response, where the infecting phage takes advantage of whatever few nutrients are available to produce small quantities of progeny within 2 to 5 h after infection. The scavenger response seems

  5. Bacteriophage T4 Infection of Stationary Phase E. coli: Life after Log from a Phage Perspective

    PubMed Central

    Bryan, Daniel; El-Shibiny, Ayman; Hobbs, Zack; Porter, Jillian; Kutter, Elizabeth M.

    2016-01-01

    Virtually all studies of phage infections investigate bacteria growing exponentially in rich media. In nature, however, phages largely encounter non-growing cells. Bacteria entering stationary phase often activate well-studied stress defense mechanisms that drastically alter the cell, facilitating its long-term survival. An understanding of phage-host interactions in such conditions is of major importance from both an ecological and therapeutic standpoint. Here, we show that bacteriophage T4 can efficiently bind to, infect and kill E. coli in stationary phase, both in the presence and absence of a functional stationary-phase sigma factor, and explore the response of T4-infected stationary phase cells to the addition of fresh nutrients 5 or 24 h after that infection. An unexpected new mode of response has been identified. “Hibernation” mode is a persistent but reversible dormant state in which the infected cells make at least some phage enzymes, but halt phage development until appropriate nutrients become available before producing phage particles. Our evidence indicates that the block in hibernation mode occurs after the middle-mode stage of phage development; host DNA breakdown and the incorporation of the released nucleotides into phage DNA indicate that the enzymes of the nucleotide synthesizing complex, under middle-mode control, have been made and assembled into a functional state. Once fresh glucose and amino acids become available, the standard lytic infection process rapidly resumes and concentrations of up to 1011 progeny phage (an average of about 40 phage per initially present cell) are produced. All evidence is consistent with the hibernation-mode control point lying between middle mode and late mode T4 gene expression. We have also observed a “scavenger” response, where the infecting phage takes advantage of whatever few nutrients are available to produce small quantities of progeny within 2 to 5 h after infection. The scavenger response seems

  6. Bacteriophage T4 Infection of Stationary Phase E. coli: Life after Log from a Phage Perspective.

    PubMed

    Bryan, Daniel; El-Shibiny, Ayman; Hobbs, Zack; Porter, Jillian; Kutter, Elizabeth M

    2016-01-01

    Virtually all studies of phage infections investigate bacteria growing exponentially in rich media. In nature, however, phages largely encounter non-growing cells. Bacteria entering stationary phase often activate well-studied stress defense mechanisms that drastically alter the cell, facilitating its long-term survival. An understanding of phage-host interactions in such conditions is of major importance from both an ecological and therapeutic standpoint. Here, we show that bacteriophage T4 can efficiently bind to, infect and kill E. coli in stationary phase, both in the presence and absence of a functional stationary-phase sigma factor, and explore the response of T4-infected stationary phase cells to the addition of fresh nutrients 5 or 24 h after that infection. An unexpected new mode of response has been identified. "Hibernation" mode is a persistent but reversible dormant state in which the infected cells make at least some phage enzymes, but halt phage development until appropriate nutrients become available before producing phage particles. Our evidence indicates that the block in hibernation mode occurs after the middle-mode stage of phage development; host DNA breakdown and the incorporation of the released nucleotides into phage DNA indicate that the enzymes of the nucleotide synthesizing complex, under middle-mode control, have been made and assembled into a functional state. Once fresh glucose and amino acids become available, the standard lytic infection process rapidly resumes and concentrations of up to 10(11) progeny phage (an average of about 40 phage per initially present cell) are produced. All evidence is consistent with the hibernation-mode control point lying between middle mode and late mode T4 gene expression. We have also observed a "scavenger" response, where the infecting phage takes advantage of whatever few nutrients are available to produce small quantities of progeny within 2 to 5 h after infection. The scavenger response seems able

  7. 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

  8. Increased expression of vascular endothelial growth factor attenuates contusion necrosis without influencing contusion edema after traumatic brain injury in rats.

    PubMed

    Tado, Masahiro; Mori, Tatsuro; Fukushima, Masamichi; Oshima, Hideki; Maeda, Takeshi; Yoshino, Atsuo; Aizawa, Shin; Katayama, Yoichi

    2014-04-01

    To clarify the role of vascular endothelial growth factor (VEGF) in the formation of contusion edema and necrosis after traumatic brain injury, we examined the time course of changes in the VEGF expression (enzyme-linked immunosorbent assay), cerebrovascular permeability (extravasation of Evans blue), and water content (dry-wet weight method) of the contused brain tissue in a cortical impact injury model using rats. In addition, we tested the effects of administration of bevacizumab (VEGF monoclonal antibody) on changes in the cerebrovascular permeability and water content of the contused brain tissue, as well as the neurological deficits (rota rod test) and volume of contusion necrosis. Increased VEGF expression was maximal at 72 h after injury (p<0.003). Increases in cerebrovascular permeability and water content, however, became maximal within 24 h (p<0.001) after injury (p<0.01), respectively. Administration of bevacizumab did not influence these changes in cerebrovascular permeability and water content, but led to a significant rise in the neurological deficits at 72 h-14 days (p<0.05 or 0.01) and the volume of contusion necrosis at 21 days (p<0.001) after injury. These findings suggest that increased expression of VEGF after injury does not contribute to the formation of contusion edema, but attenuates the formation of contusion necrosis. This is probably because of an increased angiogenesis and improved microcirculation in the areas surrounding the core of contusion. PMID:24294928

  9. 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

  10. 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

  11. Resveratrol attenuates renal injury and fibrosis by inhibiting transforming growth factor-β pathway on matrix metalloproteinase 7

    PubMed Central

    Xiao, Zhou; Chen, Chen; Meng, Ting; Zhang, Wenzheng

    2015-01-01

    Renal injury has a strong relationship to the subsequent development of renal fibrosis. In developing renal fibrosis, tubular epithelial cells in the kidney underwent epithelial–mesenchymal transition (EMT). Matrix metalloproteinase 7 (MMP7) was reported to reduce E-cadherin and induce EMT by up-regulation of β-catenin/lymphoid enhancer-binding factor 1 (LEF1) signaling. In this research, we tried to evaluate the role of resveratrol (RSV) on EMT process in renal injury and fibrosis. Human tubular epithelial cell HK-2 cells were treated with aristolochic acid (AAs) and transforming growth factor-β(TGF-β) to induce EMT with or without the administration of RSV. The inhibitory role of RSV on EMT in renal injury and fibrosis was determined by Western blotting, real-time PCR, and immunofluorescence staining. The EMT repressing role of RSV was also evaluated in vivo by renal ischemia-reperfusion (I/R) injury and unilateral ureteral obstruction (UUO) models. The underlying mechanism was investigated by shRNA interfering MMP7 and sirtuin 1 (SIRT1) expression. The results indicated that RSV reversed human kidney 2 (HK-2) cell EMT, renal I/R injury, and renal fibrosis. MMP7 inhibition was responsible for RSV-induced EMT repression. SIRT1 was up-regulated by RSV inhibited TGF-β pathway on MMP7 via deacetylating Smad4. In conclusion, RSV attenuated renal injury and fibrosis by inhibiting EMT process which was attributed to the fact that the up-regulated SIRT1 by RSV deacetylated Smad4 and inhibited MMP7 expression. PMID:26316584

  12. Resveratrol attenuates renal injury and fibrosis by inhibiting transforming growth factor-β pathway on matrix metalloproteinase 7.

    PubMed

    Xiao, Zhou; Chen, Chen; Meng, Ting; Zhang, Wenzheng; Zhou, Qiaoling

    2016-01-01

    Renal injury has a strong relationship to the subsequent development of renal fibrosis. In developing renal fibrosis, tubular epithelial cells in the kidney underwent epithelial-mesenchymal transition (EMT). Matrix metalloproteinase 7 (MMP7) was reported to reduce E-cadherin and induce EMT by up-regulation of β-catenin/lymphoid enhancer-binding factor 1 (LEF1) signaling. In this research, we tried to evaluate the role of resveratrol (RSV) on EMT process in renal injury and fibrosis. Human tubular epithelial cell HK-2 cells were treated with aristolochic acid (AAs) and transforming growth factor-β(TGF-β) to induce EMT with or without the administration of RSV. The inhibitory role of RSV on EMT in renal injury and fibrosis was determined by Western blotting, real-time PCR, and immunofluorescence staining. The EMT repressing role of RSV was also evaluated in vivo by renal ischemia-reperfusion (I/R) injury and unilateral ureteral obstruction (UUO) models. The underlying mechanism was investigated by shRNA interfering MMP7 and sirtuin 1 (SIRT1) expression. The results indicated that RSV reversed human kidney 2 (HK-2) cell EMT, renal I/R injury, and renal fibrosis. MMP7 inhibition was responsible for RSV-induced EMT repression. SIRT1 was up-regulated by RSV inhibited TGF-β pathway on MMP7 via deacetylating Smad4. In conclusion, RSV attenuated renal injury and fibrosis by inhibiting EMT process which was attributed to the fact that the up-regulated SIRT1 by RSV deacetylated Smad4 and inhibited MMP7 expression.

  13. 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

  14. Spatial variability of UVR attenuation and bio-optical factors in shallow coral-reef waters of Malaysia

    NASA Astrophysics Data System (ADS)

    Kuwahara, V. S.; Nakajima, R.; Othman, B. H. R.; Kushairi, M. R. M.; Toda, T.

    2010-09-01

    Biologically diverse coral-reef ecosystems are both directly and indirectly susceptible to changes in the spectral ultraviolet radiation (UVR) distribution. The purpose of this study was to (1) measure the variability of UVR and photosynthetically active radiation (PAR) penetration in the water above coral reefs around the Malaysian peninsula, (2) measure the variability and distribution of UVR-specific biogeochemical factors, and (3) determine the impact of biogeochemical variability as it affects the UVR:PAR ratio. Downwelling UVR and PAR irradiance and bio-optically derived biogeochemical factors were measured at 14 coral survey stations around the Malaysian peninsula from August 10-29, 2007. The West Coast was characterized by relatively shallow mean 10% UV-B (320 nm) penetration (1.68 ± 1.12 m), high chlorophyll (3.00 ± 4.72 μg l-1), high chromophoric dissolved organic matter (CDOM; 6.61 ± 3.31 ppb), high particulate organic carbon (POC; 190.65 ± 97.99 mg m-3), and low dissolved organic carbon (DOC; 1.34 ± 0.65 mg m-3). By contrast, the East Coast was characterized by relatively deep mean 10% UV-B penetration (5.03 ± 2.19 m), low chlorophyll (0.34 ± 0.22 μg l-1), low CDOM (1.45 ± 0.44 ppb), low POC (103.21 ± 37.93 mg m-3), and relatively high DOC (1.91 ± 1.03 mg m-3). The UVR:PAR ratio was relatively higher on the East Coast relative to the West Coast, suggesting variable concentrations of UVR-specific absorbing components. At all sites, UVR attenuation coefficients showed significant correlations with CDOM, but were spatially dependent with regard to chlorophyll a, POC, and DOC. The results suggest that bio-optically significant CDOM and DOC factors are uncoupled in coral-reef communities of Malaysia. Furthermore, the results support prior studies that show chromophorically active concentrations of DOM and POC are significantly altering the amount of UVR penetration above coral reefs and may be notable factors in regulating intricate

  15. Genomic characterization provides new insight into Salmonella phage diversity

    PubMed Central

    2013-01-01

    Background Salmonella is a widely distributed foodborne pathogen that causes tens of millions of salmonellosis cases globally every year. While the genomic diversity of Salmonella is increasingly well studied, our knowledge of Salmonella phage genomic diversity is still rather limited, despite the contributions of both lysogenic and lytic phages to Salmonella virulence, diversity and ecology (e.g., through horizontal gene transfer and Salmonella lysis). To gain a better understanding of phage diversity in a specific ecological niche, we sequenced 22 Salmonella phages isolated from a number of dairy farms from New York State (United States) and analyzed them using a comparative genomics approach. Results Classification of the 22 phages according to the presence/absence of orthologous genes allowed for classification into 8 well supported clusters. In addition to two phage clusters that represent novel virulent Salmonella phages, we also identified four phage clusters that each contained previously characterized phages from multiple continents. Our analyses also identified two clusters of phages that carry putative virulence (e.g., adhesins) and antimicrobial resistance (tellurite and bicyclomycin) genes as well as virulent and temperate transducing phages. Insights into phage evolution from our analyses include (i) identification of DNA metabolism genes that may facilitate nucleotide synthesis in phages with a G+C % distinct from Salmonella, and (ii) evidence of Salmonella phage tailspike and fiber diversity due to both single nucleotide polymorphisms and major re-arrangements, which may affect the host specificity of Salmonella phages. Conclusions Genomics-based characterization of 22 Salmonella phages isolated from dairy farms allowed for identification of a number of novel Salmonella phages. While the comparative genomics analyses of these phages provide a number of new insights in the evolution and diversity of Salmonella phages, they only represent a first

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. 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

  18. 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

  19. Sad State of Phage Electron Microscopy. Please Shoot the Messenger

    PubMed Central

    Ackermann, Hans-W.

    2013-01-01

    Two hundred and sixty publications from 2007 to 2012 were classified according to the quality of electron micrographs; namely as good (71); mediocre (21); or poor (168). Publications were from 37 countries; appeared in 77 journals; and included micrographs produced with about 60 models of electron microscopes. The quality of the micrographs was not linked to any country; journal; or electron microscope. Main problems were poor contrast; positive staining; low magnification; and small image size. Unsharp images were frequent. Many phage descriptions were silent on virus purification; magnification control; even the type of electron microscope and stain used. The deterioration in phage electron microscopy can be attributed to the absence of working instructions and electron microscopy courses; incompetent authors and reviewers; and lenient journals. All these factors are able to cause a gradual lowering of standards.

  20. 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

  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. 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

  3. Rapid enumeration of phage in monodisperse emulsions.

    PubMed

    Tjhung, Katrina F; Burnham, Sean; Anany, Hany; Griffiths, Mansel W; Derda, Ratmir

    2014-06-17

    Phage-based detection assays have been developed for the detection of viable bacteria for applications in clinical diagnosis, monitoring of water quality, and food safety. The majority of these assays deliver a positive readout in the form of newly generated progeny phages by the bacterial host of interest. Progeny phages are often visualized as plaques, or holes, in a lawn of bacteria on an agar-filled Petri dish; however, this rate-limiting step requires up to 12 h of incubation time. We have previously described an amplification of bacteriophages M13 inside droplets of media suspended in perfluorinated oil; a single phage M13 in a droplet yields 10(7) copies in 3-4 h. Here, we describe that encapsulation of reporter phages, both lytic T4-LacZ and nonlytic M13, in monodisperse droplets can also be used for rapid enumeration of phage. Compartmentalization in droplets accelerated the development of the signal from the reporter enzyme; counting of "positive" droplets yields accurate enumeration of phage particles ranging from 10(2) to 10(6) pfu/mL. For enumeration of T4-LacZ phage, the fluorescent signal appeared in as little as 90 min. Unlike bulk assays, quantification in emulsion is robust and insensitive to fluctuations in environmental conditions (e.g., temperature). Power-free emulsification using gravity-driven flow in the absence of syringe pumps and portable fluorescence imaging solutions makes this technology promising for use at the point of care in low-resource environments. This droplet-based phage enumeration method could accelerate and simplify point-of-care detection of the pathogens for which reporter bacteriophages have been developed.

  4. Rapid enumeration of phage in monodisperse emulsions.

    PubMed

    Tjhung, Katrina F; Burnham, Sean; Anany, Hany; Griffiths, Mansel W; Derda, Ratmir

    2014-06-17

    Phage-based detection assays have been developed for the detection of viable bacteria for applications in clinical diagnosis, monitoring of water quality, and food safety. The majority of these assays deliver a positive readout in the form of newly generated progeny phages by the bacterial host of interest. Progeny phages are often visualized as plaques, or holes, in a lawn of bacteria on an agar-filled Petri dish; however, this rate-limiting step requires up to 12 h of incubation time. We have previously described an amplification of bacteriophages M13 inside droplets of media suspended in perfluorinated oil; a single phage M13 in a droplet yields 10(7) copies in 3-4 h. Here, we describe that encapsulation of reporter phages, both lytic T4-LacZ and nonlytic M13, in monodisperse droplets can also be used for rapid enumeration of phage. Compartmentalization in droplets accelerated the development of the signal from the reporter enzyme; counting of "positive" droplets yields accurate enumeration of phage particles ranging from 10(2) to 10(6) pfu/mL. For enumeration of T4-LacZ phage, the fluorescent signal appeared in as little as 90 min. Unlike bulk assays, quantification in emulsion is robust and insensitive to fluctuations in environmental conditions (e.g., temperature). Power-free emulsification using gravity-driven flow in the absence of syringe pumps and portable fluorescence imaging solutions makes this technology promising for use at the point of care in low-resource environments. This droplet-based phage enumeration method could accelerate and simplify point-of-care detection of the pathogens for which reporter bacteriophages have been developed. PMID:24892245

  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. 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

  7. 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

  8. Connective tissue growth factor inhibition attenuates left ventricular remodeling and dysfunction in pressure overload-induced heart failure.

    PubMed

    Szabó, Zoltán; Magga, Johanna; Alakoski, Tarja; Ulvila, Johanna; Piuhola, Jarkko; Vainio, Laura; Kivirikko, Kari I; Vuolteenaho, Olli; Ruskoaho, Heikki; Lipson, Kenneth E; Signore, Pierre; Kerkelä, Risto

    2014-06-01

    Connective tissue growth factor (CTGF) is involved in the pathogenesis of various fibrotic disorders. However, its role in the heart is not clear. To investigate the role of CTGF in regulating the development of cardiac fibrosis and heart failure, we subjected mice to thoracic aortic constriction (TAC) or angiotensin II infusion, and antagonized the function of CTGF with CTGF monoclonal antibody (mAb). After 8 weeks of TAC, mice treated with CTGF mAb had significantly better preserved left ventricular (LV) systolic function and reduced LV dilatation compared with mice treated with control immunoglobulin G. CTGF mAb-treated mice exhibited significantly smaller cardiomyocyte cross-sectional area and reduced expression of hypertrophic marker genes. CTGF mAb treatment reduced the TAC-induced production of collagen 1 but did not significantly attenuate TAC-induced accumulation of interstitial fibrosis. Analysis of genes regulating extracellular matrix proteolysis showed decreased expression of plasminogen activator inhibitor-1 and matrix metalloproteinase-2 in mice treated with CTGF mAb. In contrast to TAC, antagonizing the function of CTGF had no effect on LV dysfunction or LV hypertrophy in mice subjected to 4-week angiotensin II infusion. Further analysis showed that angiotensin II-induced expression of hypertrophic marker genes or collagens was not affected by treatment with CTGF mAb. In conclusion, CTGF mAb protects from adverse LV remodeling and LV dysfunction in hearts subjected to pressure overload by TAC. Antagonizing the function of CTGF may offer protection from cardiac end-organ damage in patients with hypertension.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. Platelet-activating factor antagonist TCV-309 attenuates the induction of the cytokine network in experimental endotoxemia in chimpanzees.

    PubMed

    Kuipers, B; van der Poll, T; Levi, M; van Deventer, S J; ten Cate, H; Imai, Y; Hack, C E; ten Cate, J W

    1994-03-01

    Platelet-activating factor (PAF) has been postulated to play a role in the pathogenesis of sepsis. Additionally, in vitro studies have revealed tight interactions between PAF and the cytokine network, and PAF is considered to be an important stimulator of neutrophil functions. To assess the intermediate role of PAF in the induction of cytokines and neutrophil degranulation in endotoxemia in vivo, 12 healthy adult chimpanzees were i.v. injected with a bolus dose of Escherichia coli endotoxin (4 ng/kg); four animals received endotoxin alone, whereas the other chimpanzees were infused with the specific and potent PAF antagonist TCV-309 (bolus of 100 micrograms/kg, followed by either 100 micrograms/kg/h (n = 4) or 500 micrograms/kg/h (n = 4) for 5 h). At both doses TCV-309 significantly inhibited the endotoxin-induced rise in cytokine levels. Peak TNF concentrations after injection of endotoxin alone were 366 +/- 96 pg/ml, vs 105 +/- 47 and 115 +/- 56 pg/ml after administration of endotoxin together with the lower or higher dose of TCV-309, respectively (p < 0.05). TCV-309 also reduced the appearance of soluble TNFRs. Maximal levels of the type I soluble TNFR were diminished from 2.53 +/- 0.27 ng/ml (endotoxin alone) to 1.69 +/- 0.36 ng/ml (high dose TCV-309; p < 0.05); peak values of the type II soluble TNFR were diminished from 8.62 +/- 1.19 ng/ml to 5.76 +/- 0.92 ng/ml (p < 0.05). Furthermore, TCV-309 attenuated the endotoxin-induced release of IL-6 (160 +/- 82 pg/ml after endotoxin alone, vs 63 +/- 30 pg/ml in the low dose TCV-309 group (p < 0.05) and 65 +/- 29 pg/ml in the high dose group (p = 0.07) as well as that of IL-8 (279 +/- 168, vs 71 +/- 15 and 46 +/- 17 pg/ml, respectively; both p < 0.05). TCV-309 tended to reduce the endotoxin-provoked rise in serum IL-1R antagonist levels. In contrast, TCV-309 did not affect the neutrophilic leukocytosis elicited by endotoxin, nor did it inhibit endotoxin-induced neutrophil degranulation, as monitored by the plasma

  16. 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

  17. PLANET: a phage library analysis expert tool.

    PubMed

    Leplae, R; Tramontano, A

    1995-01-01

    In recent years random peptide libraries displayed on filamentous phage have been widely used and new ideas and techniques are continuously developing in the field (1-5). Notwithstanding this growing interest in the technique and in its promising results, and the enormous increase in usage and scope, very little effort has been devoted to the implementation of software able to handle and analyze the growing number of phage library-derived sequences. In our laboratory, phage libraries are extensively used and peptide sequences are continuously produced, so that the need arose of creating a database (6) to collect all the experimental results in a format compatible with GCG sequence analysis packages (7). We present here the description of an XWindow-based software package named PLANET (Phage Library ANalysis Expert Tool) devoted to the maintenance and statistical analysis of the database.

  18. Phage therapy treatment of the coral pathogen Vibrio coralliilyticus

    PubMed Central

    Cohen, Yossi; Joseph Pollock, F; Rosenberg, Eugene; Bourne, David G

    2013-01-01

    Vibrio coralliilyticus is an important coral pathogen demonstrated to cause disease outbreaks worldwide. This study investigated the feasibility of applying bacteriophage therapy to treat the coral pathogen V. coralliilyticus. A specific bacteriophage for V. coralliilyticus strain P1 (LMG23696), referred to here as bacteriophage YC, was isolated from the seawater above corals at Nelly Bay, Magnetic Island, central Great Barrier Reef (GBR), the same location where the bacterium was first isolated. Bacteriophage YC was shown to be a lytic phage belonging to the Myoviridae family, with a rapid replication rate, high burst size, and high affinity to its host. By infecting its host bacterium, bacteriophage YC was able to prevent bacterial-induced photosystem inhibition in pure cultures of Symbiodinium, the photosymbiont partner of coral and a target for virulence factors produced by the bacterial pathogen. Phage therapy experiments using coral juveniles in microtiter plates as a model system revealed that bacteriophage YC was able to prevent V. coralliilyticus-induced photoinactivation and tissue lysis. These results demonstrate that bacteriophage YC has the potential to treat coral disease outbreaks caused by the bacterial pathogen V. coralliilyticus, making it a good candidate for phage therapy treatment of coral disease. PMID:23239510

  19. 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

  20. 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.

  1. Chronic Sleep Restriction Elevates Brain Interleukin-1 beta and Tumor Necrosis Factor-alpha and Attenuates Brain-derived Neurotrophic Factor Expression

    PubMed Central

    Zielinski, Mark R.; Kim, Youngsoo; Karpova, Svetlana A.; McCarley, Robert W.; Strecker, Robert E.; Gerashchenko, Dmitry

    2014-01-01

    Acute sleep loss increases pro-inflammatory and synaptic plasticity-related molecules in the brain, including interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and brain-derived neurotrophic factor (BDNF). These molecules enhance non-rapid eye movement sleep slow wave activity (SWA), also known as electroencephalogram delta power, and modulate neurocognitive performance. Evidence suggests that chronic sleep restriction (CSR), a condition prevalent in today's society, does not elicit the enhanced SWA that is seen after acute sleep loss, although it cumulatively impairs neurocognitive functioning. Rats were continuously sleep deprived for 18 h per day and allowed 6 h of ad libitum sleep opportunity for 1 (SR1), 3 (SR3), or 5 (SR5) successive days (i.e., CSR). IL-1β, TNF-α, and BDNF mRNA levels were determined in the somatosensory cortex, frontal cortex, hippocampus, and basal forebrain. Largely, brain IL-1β and TNF-α expression were significantly enhanced throughout CSR. In contrast, BDNF mRNA levels were similar to baseline values in the cortex after 1 day of SR and significantly lower than baseline values in the hippocampus after 5 days of SR. In the basal forebrain, BDNF expression remained elevated throughout the 5 days of CSR, although IL-1β expression was significantly reduced. The chronic elevations of IL-1β and TNF-α and inhibition of BDNF might contribute to the reported lack of SWA responses reported after CSR. Further, the CSR-induced enhancements in brain inflammatory molecules and attenuations in hippocampal BDNF might contribute to neurocognitive and vigilance detriments that occur from CSR. PMID:25093703

  2. Chronic sleep restriction elevates brain interleukin-1 beta and tumor necrosis factor-alpha and attenuates brain-derived neurotrophic factor expression.

    PubMed

    Zielinski, Mark R; Kim, Youngsoo; Karpova, Svetlana A; McCarley, Robert W; Strecker, Robert E; Gerashchenko, Dmitry

    2014-09-19

    Acute sleep loss increases pro-inflammatory and synaptic plasticity-related molecules in the brain, including interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and brain-derived neurotrophic factor (BDNF). These molecules enhance non-rapid eye movement sleep slow wave activity (SWA), also known as electroencephalogram delta power, and modulate neurocognitive performance. Evidence suggests that chronic sleep restriction (CSR), a condition prevalent in today's society, does not elicit the enhanced SWA that is seen after acute sleep loss, although it cumulatively impairs neurocognitive functioning. Rats were continuously sleep deprived for 18h per day and allowed 6h of ad libitum sleep opportunity for 1 (SR1), 3 (SR3), or 5 (SR5) successive days (i.e., CSR). IL-1β, TNF-α, and BDNF mRNA levels were determined in the somatosensory cortex, frontal cortex, hippocampus, and basal forebrain. Largely, brain IL-1β and TNF-α expression were significantly enhanced throughout CSR. In contrast, BDNF mRNA levels were similar to baseline values in the cortex after 1 day of SR and significantly lower than baseline values in the hippocampus after 5 days of SR. In the basal forebrain, BDNF expression remained elevated throughout the 5 days of CSR, although IL-1β expression was significantly reduced. The chronic elevations of IL-1β and TNF-α and inhibition of BDNF might contribute to the reported lack of SWA responses reported after CSR. Further, the CSR-induced enhancements in brain inflammatory molecules and attenuations in hippocampal BDNF might contribute to neurocognitive and vigilance detriments that occur from CSR.

  3. 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.

  4. 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

  5. BLOCKADE OF NERVE GROWTH FACTOR (NGF) RECEPTOR TRKA ATTENUATES DIESEL EXHAUST PARTICULATE MATTER (DEP) ENHANCEMENT OF ALLERGIC INFLAMMATION

    EPA Science Inventory


    Recent studies have shown that asthmatics have increased levels of the neurotrophin, NGF, in their lungs. In addition, antibody blockade of NGF in mice attenuates airway resistance associated with allergic airway responses. DEP has been linked to asthma exacerbation in many c...

  6. Bacteriophage 2851 Is a Prototype Phage for Dissemination of the Shiga Toxin Variant Gene 2c in Escherichia coli O157:H7▿ †

    PubMed Central

    Strauch, Eckhard; Hammerl, Jens Andre; Konietzny, Antje; Schneiker-Bekel, Susanne; Arnold, Walter; Goesmann, Alexander; Pühler, Alfred; Beutin, Lothar

    2008-01-01

    The production of Shiga toxin (Stx) (verocytotoxin) is a major virulence factor of Escherichia coli O157:H7 strains (Shiga toxin-producing E. coli [STEC] O157). Two types of Shiga toxins, designated Stx1 and Stx2, are produced in STEC O157. Variants of the Stx2 type (Stx2, Stx2c) are associated with high virulences of these strains for humans. A bacteriophage designated 2851 from a human STEC O157 encoding the Stx2c variant was described previously. Nucleotide sequence analysis of the phage 2851 genome revealed 75 predicted coding sequences and indicated a mosaic structure typical for lambdoid phages. Analyses of free phages and K-12 phage 2851 lysogens revealed that upon excision from the bacterial chromosome, the loss of a phage-encoded IS629 element leads to fusion of phage antA and antB genes, with the generation of a recombined antAB gene encoding a strong antirepressor. In wild-type E. coli O157 as well as in K-12 strains, phage 2851 was found to be integrated in the sbcB locus. Additionally, phage 2851 carries an open reading frame which encodes an OspB-like type III effector similar to that found in Shigella spp. Investigation of 39 stx2c E. coli O157 strains revealed that all except 1 were positive for most phage 2851-specific genes and possessed a prophage with the same border sequences integrated into the sbcB locus. Phage 2851-specific sequences were absent from most stx2c-negative E. coli O157 strains, and we suggest that phage 2851-like phages contributed significantly to the dissemination of the Stx2c variant toxin within this group of E. coli. PMID:18824528

  7. 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.

  8. [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

  9. The production of generalized transducing phage by bacteriophage lambda.

    PubMed

    Sternberg, N

    1986-01-01

    Generalized transduction has for about 30 years been a major tool in the genetic manipulation of bacterial chromosomes. However, throughout that time little progress has been made in understanding how generalized transducing particles are produced. The experiments presented in this paper use phage lambda to assess some of the factors that affect that process. The results of those experiments indicate: the production of generalized transducing particles by bacteriophage lambda is inhibited by the phage lambda exonuclease (Exo). Also inhibited by lambda Exo is the production of lambda docR particles, a class of particles whose packaging is initiated in bacterial DNA and terminated at the normal phage packaging site, cos. In contrast, the production of lambda docL particles, a class of particles whose packaging is initiated at cos and terminated in bacterial DNA, is unaffected by lambda Exo; lambda-generalized transducing particles are not detected in induced lysis-defective (S-) lambda lysogens until about 60-90 min after prophage induction. Since wild-type lambda would normally lyse cells by 60 min, the production of lambda-generalized transducing particles depends on the phage being lysis-defective; if transducing lysates are prepared by phage infection then the frequency of generalized transduction for different bacterial markers varies over a 10-20-fold range. In contrast, if transducing lysates are prepared by the induction of a lambda lysogen containing an excision-defective prophage, then the variation in transduction frequency is much greater, and markers adjacent to, and on both sides of, the prophage are transduced with much higher frequencies than are other markers; if the prophage is replication-defective then the increased transduction of prophage-proximal markers is eliminated; measurements of total DNA in induced lysogens indicate that part of the increase in transduction frequency following prophage induction can be accounted for by an increase in the

  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. Antibody Phage Display Libraries: Contributions to Oncology

    PubMed Central

    Dantas-Barbosa, Carmela; de Macedo Brigido, Marcelo; Maranhao, Andrea Queiroz

    2012-01-01

    Since the advent of phage display technology, dating back to 1985, antibody libraries displayed on filamentous phage surfaces have been used to identify specific binders for many different purposes, including the recognition of tumors. Phage display represents a high-throughput technique for screening billions of random fusion antibodies against virtually any target on the surface or inside cancer cells, or even soluble markers found in patient serum. Many phage display derived binders targeting important tumor markers have been identified. Selection directed to tumoral cells’ surfaces lead to the identification of unknown tumoral markers. Also the improvement of methods that require smaller amounts of cells has opened the possibility to use this approach on patient samples. Robust techniques combining an antibody library displayed on the phage surface and protein microarray allowed the identification of auto antibodies recognized by patient sera. Many Ab molecules directly or indirectly targeting angiogenesis have been identified, and one of them, ramucirumab, has been tested in 27 phase I–III clinical trials in a broad array of cancers. Examples of such antibodies will be discussed here with emphasis on those used as probes for molecular imaging and other clinical trials. PMID:22754305

  12. 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.

  13. Phage display of engineered binding proteins.

    PubMed

    Levisson, Mark; Spruijt, Ruud B; Winkel, Ingrid Nolla; Kengen, Servé W M; van der Oost, John

    2014-01-01

    In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.

  14. Network models of phage-bacteria coevolution

    NASA Astrophysics Data System (ADS)

    Rosvall, Martin; Dodd, Ian B.; Krishna, Sandeep; Sneppen, Kim

    2006-12-01

    Bacteria and their bacteriophages are the most abundant, widespread, and diverse groups of biological entities on the planet. In an attempt to understand how the interactions between bacteria, virulent phages, and temperate phages might affect the diversity of these groups, we developed a stochastic network model for examining the coevolution of these ecologies. In our approach, nodes represent whole species or strains of bacteria or phages, rather than individuals, with “speciation” and extinction modeled by duplication and removal of nodes. Phage-bacteria links represent host-parasite relationships and temperate-virulent phage links denote prophage-encoded resistance. The effect of horizontal transfer of genetic information between strains was also included in the dynamical rules. The observed networks evolved in a highly dynamic fashion but the ecosystems were prone to collapse (one or more entire groups going extinct). Diversity could be stably maintained in the model only if the probability of speciation was independent of the diversity. Such an effect could be achieved in real ecosystems if the speciation rate is primarily set by the availability of ecological niches.

  15. T-cell factor-4 and MHC upregulation in pigs receiving a live attenuated classical swine fever virus (CSFV) vaccine strain with interferon-gamma adjuvant.

    PubMed

    Fan, Y-H; Lin, Y-L; Hwang, Y-C; Yang, H-C; Chiu, H-C; Chiou, S-H; Jong, M-H; Chow, K-C; Lin, C-C

    2016-10-01

    The effect of co-administration of interferon (IFN)-γ in pigs undergoing vaccination with an attenuated strain (LPC) of classical swine fever virus (CSFV) was investigated. Unvaccinated pigs demonstrated pyrexia and died 7-9 days after challenge with virulent CSFV. Pigs receiving the attenuated vaccine remained healthy after virus challenge, except for mild, transient pyrexia, whereas pigs receiving IFN-γ simultaneously with the vaccine demonstrated normal body temperatures after virus challenge. Examination by nested RT-PCR revealed greater viral load in the spleens of the pigs vaccinated with the attenuated CSFV, compared with those that had additionally received IFN-γ. Expression of major histocompatibility complex (MHC) class I and MHC class II molecules was upregulated in the spleens of the IFN-γ treated vaccinated pigs, demonstrated by immunohistochemistry. Based on Western blot analysis, anti-CSFV IgG2 antibodies were elevated in vaccinated pigs by co-administration of IFN-γ (IFN-γ(Hi): P < 0.01; IFN-γ(Lo): P <0.05). By employing the suppression subtractive hybridization technique, RT-PCR, in situ hybridization, and immunohistochemistry, T-cell factor-4 (Tcf-4) mRNA and protein expression were found to be upregulated in the spleens of vaccinated pigs that had received IFN-γ. This study suggests involvement of Tcf-4 in IFN-γ-mediated immune regulation following CSFV vaccination. PMID:27687943

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. Propagation method for persistent high yield of diverse Listeria phages on permissive hosts at refrigeration temperatures.

    PubMed

    Radford, Devon R; Ahmadi, Hanie; Leon-Velarde, Carlos G; Balamurugan, Sampathkumar

    2016-10-01

    The efficient production of a high concentration of bacteriophage in large volumes has been a limiting factor in the exploration of the true potential of these organisms for biotechnology, agriculture and medicine. Traditional methods focus on generating small volumes of highly concentrated samples as the end product of extensive mechanical and osmotic processing. To function at an industrial scale mandates extensive investment in infrastructure and input materials not feasible for many smaller facilities. To address this, we developed a novel, scalable, generic method for producing significantly higher titer psychrophilic phage (P < 2.0 × 10(-6)), 2- to 4-fold faster than traditional methods. We generate renewable high yields from single source cultures by propagating phage under refrigeration conditions in which Listeria, Yersinia and their phages grow in equilibrium. Diverse Yersinia and Listeria phages tested yielded averages of 3.49 × 10(8) to 3.36 × 10(12) PFU/ml/day compared to averages of 1.28 × 10(5) to 1.30 × 10(10) PFU/ml/day by traditional methods. Host growth and death kinetics made this method ineffective for extended propagation of mesophilic phages.

  3. Propagation method for persistent high yield of diverse Listeria phages on permissive hosts at refrigeration temperatures.

    PubMed

    Radford, Devon R; Ahmadi, Hanie; Leon-Velarde, Carlos G; Balamurugan, Sampathkumar

    2016-10-01

    The efficient production of a high concentration of bacteriophage in large volumes has been a limiting factor in the exploration of the true potential of these organisms for biotechnology, agriculture and medicine. Traditional methods focus on generating small volumes of highly concentrated samples as the end product of extensive mechanical and osmotic processing. To function at an industrial scale mandates extensive investment in infrastructure and input materials not feasible for many smaller facilities. To address this, we developed a novel, scalable, generic method for producing significantly higher titer psychrophilic phage (P < 2.0 × 10(-6)), 2- to 4-fold faster than traditional methods. We generate renewable high yields from single source cultures by propagating phage under refrigeration conditions in which Listeria, Yersinia and their phages grow in equilibrium. Diverse Yersinia and Listeria phages tested yielded averages of 3.49 × 10(8) to 3.36 × 10(12) PFU/ml/day compared to averages of 1.28 × 10(5) to 1.30 × 10(10) PFU/ml/day by traditional methods. Host growth and death kinetics made this method ineffective for extended propagation of mesophilic phages. PMID:27287043

  4. Bacteria between protists and phages: from antipredation strategies to the evolution of pathogenicity.

    PubMed

    Brüssow, Harald

    2007-08-01

    Bacteriophages and protists are major causes of bacterial mortality. Genomics suggests that phages evolved well before eukaryotic protists. Bacteria were thus initially only confronted with phage predators. When protists evolved, bacteria were caught between two types of predators. One successful antigrazing strategy of bacteria was the elaboration of toxins that would kill the grazer. The released cell content would feed bystander bacteria. I suggest here that, to fight grazing protists, bacteria teamed up with those phage predators that concluded at least a temporary truce with them in the form of lysogeny. Lysogeny was perhaps initially a resource management strategy of phages that could not maintain infection chains. Subsequently, lysogeny might have evolved into a bacterium-prophage coalition attacking protists, which became a food source for them. When protists evolved into multicellular animals, the lysogenic bacteria tracked their evolving food source. This hypothesis could explain why a frequent scheme of bacterial pathogenicity is the survival in phagocytes, why a significant fraction of bacterial pathogens have prophage-encoded virulence genes, and why some virulence factors of animal pathogens are active against unicellular eukaryotes. Bacterial pathogenicity might thus be one playing option of the stone-scissor-paper game played between phages-bacteria-protists, with humans getting into the crossfire.

  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. The Caulobacter crescentus phage phiCbK: genomics of a canonical phage

    PubMed Central

    2012-01-01

    Background The bacterium Caulobacter crescentus is a popular model for the study of cell cycle regulation and senescence. The large prolate siphophage phiCbK has been an important tool in C. crescentus biology, and has been studied in its own right as a model for viral morphogenesis. Although a system of some interest, to date little genomic information is available on phiCbK or its relatives. Results Five novel phiCbK-like C. crescentus bacteriophages, CcrMagneto, CcrSwift, CcrKarma, CcrRogue and CcrColossus, were isolated from the environment. The genomes of phage phiCbK and these five environmental phage isolates were obtained by 454 pyrosequencing. The phiCbK-like phage genomes range in size from 205 kb encoding 318 proteins (phiCbK) to 280 kb encoding 448 proteins (CcrColossus), and were found to contain nonpermuted terminal redundancies of 10 to 17 kb. A novel method of terminal ligation was developed to map genomic termini, which confirmed termini predicted by coverage analysis. This suggests that sequence coverage discontinuities may be useable as predictors of genomic termini in phage genomes. Genomic modules encoding virion morphogenesis, lysis and DNA replication proteins were identified. The phiCbK-like phages were also found to encode a number of intriguing proteins; all contain a clearly T7-like DNA polymerase, and five of the six encode a possible homolog of the C. crescentus cell cycle regulator GcrA, which may allow the phage to alter the host cell’s replicative state. The structural proteome of phage phiCbK was determined, identifying the portal, major and minor capsid proteins, the tail tape measure and possible tail fiber proteins. All six phage genomes are clearly related; phiCbK, CcrMagneto, CcrSwift, CcrKarma and CcrRogue form a group related at the DNA level, while CcrColossus is more diverged but retains significant similarity at the protein level. Conclusions Due to their lack of any apparent relationship to other described phages, this

  8. In vivo administration of epidermal growth factor and its homologue attenuates developmental maturation of functional excitatory synapses in cortical GABAergic neurons.

    PubMed

    Nagano, Tadasato; Namba, Hisaaki; Abe, Yuichi; Aoki, Hiroyuki; Takei, Nobuyuki; Nawa, Hiroyuki

    2007-01-01

    The ErbB1 ligand family includes epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), heparin-binding EGF-like growth factor, amphiregulin and betacellulin. Previously, we demonstrated that TGFalpha decreases alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors in cultured neocortical gamma-aminobutyric acid (GABA) neurons. In the present study, we examined in vivo effects of EGF and TGFalpha in the mouse neocortex using electrophysiological and biochemical techniques. In mouse neonates, subcutaneously administered EGF penetrated the blood-brain barrier and activated ErbB1 in the neocortex. Daily administration of EGF or TGFalpha attenuates developmental increases in expression of the AMPA receptor subunits (GluR1 and GluR2/3) in the neocortex of postnatal mice. Immunohistochemistry revealed that the reduction in AMPA receptor expression was significant in the GABAergic neurons, especially those positive for parvalbumin. Using cortical slices prepared from EGF-treated mice, we recorded miniature excitatory postsynaptic currents (mEPSCs) in both GABAergic and pyramidal neurons. Subchronic treatment with EGF decreased the amplitude and frequency of mEPSCs in GABAergic neurons, but its effects were negligible on pyramidal neurons. We conclude that EGF or other ErbB1 ligand(s) attenuates a developmental increase in AMPA receptor expression and function in cortical GABAergic neurons.

  9. HostPhinder: A Phage Host Prediction Tool.

    PubMed

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

    2016-05-04

    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].

  10. 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

  11. 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

  12. Genome Sequences of Gordonia Phages Bowser and Schwabeltier.

    PubMed

    Montgomery, Matthew T; Pope, Welkin H; 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. 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

  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. Vi I typing phage for generalized transduction of Salmonella typhi.

    PubMed

    Cerquetti, M C; Hooke, A M

    1993-08-01

    Salmonella typhi Vi typing phages were used to transduce temperature-sensitive (Ts) mutants of Salmonella typhi. Antibiotic resistance and Ts+ markers were transduced at high frequency (> 10(-4) per virulent phage). Several markers were cotransduced by phage Vi I, suggesting that it may be useful for mapping studies of the S. typhi genome.

  16. 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

  17. Cell biology perspectives in phage biology.

    PubMed

    Ansaldi, Mireille

    2012-01-01

    Cellular biology has long been restricted to large cellular organisms. However, as the resolution of microscopic methods increased, it became possible to study smaller cells, in particular bacterial cells. Bacteriophage biology is one aspect of bacterial cell biology that has recently gained insight from cell biology. Despite their small size, bacteriophages could be successfully labeled and their cycle studied in the host cells. This review aims to put together, although non-extensively, several cell biology studies that recently pushed the elucidation of key mechanisms in phage biology, such as the lysis-lysogeny decision in temperate phages or genome replication and transcription, one step further.

  18. Vibrio cholerae phage K139: complete genome sequence and comparative genomics of related phages.

    PubMed

    Kapfhammer, Dagmar; Blass, Julia; Evers, Stefan; Reidl, Joachim

    2002-12-01

    In this report, we characterize the complete genome sequence of the temperate phage K139, which morphologically belongs to the Myoviridae phage family (P2 and 186). The prophage genome consists of 33,106 bp, and the overall GC content is 48.9%. Forty-four open reading frames were identified. Homology analysis and motif search were used to assign possible functions for the genes, revealing a close relationship to P2-like phages. By Southern blot screening of a Vibrio cholerae strain collection, two highly K139-related phage sequences were detected in non-O1, non-O139 strains. Combinatorial PCR analysis revealed almost identical genome organizations. One region of variable gene content was identified and sequenced. Additionally, the tail fiber genes were analyzed, leading to the identification of putative host-specific sequence variations. Furthermore, a K139-encoded Dam methyltransferase was characterized.

  19. 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

  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.

  1. Improved estimation of P-wave velocity, S-wave velocity, and attenuation factor by iterative structural joint inversion of crosswell seismic data

    NASA Astrophysics Data System (ADS)

    Zhu, Tieyuan; Harris, Jerry M.

    2015-12-01

    We present an iterative joint inversion approach for improving the consistence of estimated P-wave velocity, S-wave velocity and attenuation factor models. This type of inversion scheme links two or more independent inversions using a joint constraint, which is constructed by the cross-gradient function in this paper. The primary advantages of this joint inversion strategy are: avoiding weighting for different datasets in conventional simultaneous joint inversion, flexible for incorporating prior information, and relatively easy to code. We demonstrate the algorithm with two synthetic examples and two field datasets. The inversions for P- and S-wave velocity are based on ray traveltime tomography. The results of the first synthetic example show that the iterative joint inversion take advantages of both P- and S-wave sensitivity to resolve their ambiguities as well as improve structural similarity between P- and S-wave velocity models. In the second synthetic and field examples, joint inversion of P- and S-wave traveltimes results in an improved Vs velocity model that shows better structural correlation with the Vp model. More importantly, the resultant VP/VS ratio map has fewer artifacts and is better correlated for use in geological interpretation than the independent inversions. The second field example illustrates that the flexible joint inversion algorithm using frequency-shift data gives a structurally improved attenuation factor map constrained by a prior VP tomogram.

  2. 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

  3. 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

  4. 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

  5. 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

  6. Comparative phage genomics and the evolution of Siphoviridae: insights from dairy phages.

    PubMed

    Brüssow, H; Desiere, F

    2001-01-01

    Comparative phage genomics can retrace part of the evolutionary history of phage modules encoding phage-specific functions such as capsid building or establishment of the lysogenic state. The diagnosis of relatedness is not based exclusively on sequence similarity, but includes topological considerations of genome organization. The gene maps from the lambda-, psiM2-, L5-, Sfi21-, Sfi11-, phiC31-, sk1- and TM4-like phages showed a remarkable synteny of their structural genes defining a lambda supergroup within Siphoviridae (Caudovirales with long non-contractile tails). A hierarchy of relatedness within the lambda supergroup suggested elements of vertical evolution in the capsid module of Siphoviridae. Links to P22-like Podoviridae and P2-like Myoviridae were also detected. Numerous cases of horizontal gene transfer were observed, but recent transfers were limited to interbreeding phage populations. We suggest that tailed phages are the result of both vertical and horizontal evolution and are thus a good model system for web-like phylogenies.

  7. 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

  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.

  9. Heterogeneity in Induction Level, Infection Ability, and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates

    PubMed Central

    Bonanno, Ludivine; Petit, Marie-Agnès; Loukiadis, Estelle; Michel, Valérie

    2016-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are foodborne pathogens responsible for diarrhea and hemolytic-uremic syndrome (HUS). Shiga toxin, the main STEC virulence factor, is encoded by the stx gene located in the genome of a bacteriophage inserted into the bacterial chromosome. The O26:H11 serotype is considered to be the second-most-significant HUS-causing serotype worldwide after O157:H7. STEC O26:H11 bacteria and their stx-negative counterparts have been detected in dairy products. They may convert from the one form to the other by loss or acquisition of Stx phages, potentially confounding food microbiological diagnostic methods based on stx gene detection. Here we investigated the diversity and mobility of Stx phages from human and dairy STEC O26:H11 strains. Evaluation of their rate of in vitro induction, occurring either spontaneously or in the presence of mitomycin C, showed that the Stx2 phages were more inducible overall than Stx1 phages. However, no correlation was found between the Stx phage levels produced and the origin of the strains tested or the phage insertion sites. Morphological analysis by electron microscopy showed that Stx phages from STEC O26:H11 displayed various shapes that were unrelated to Stx1 or Stx2 types. Finally, the levels of sensitivity of stx-negative E. coli O26:H11 to six Stx phages differed among the 17 strains tested and our attempts to convert them into STEC were unsuccessful, indicating that their lysogenization was a rare event. PMID:26826235

  10. Heterogeneity in Induction Level, Infection Ability, and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates.

    PubMed

    Bonanno, Ludivine; Petit, Marie-Agnès; Loukiadis, Estelle; Michel, Valérie; Auvray, Frédéric

    2016-01-29

    Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are foodborne pathogens responsible for diarrhea and hemolytic-uremic syndrome (HUS). Shiga toxin, the main STEC virulence factor, is encoded by the stx gene located in the genome of a bacteriophage inserted into the bacterial chromosome. The O26:H11 serotype is considered to be the second-most-significant HUS-causing serotype worldwide after O157:H7. STEC O26:H11 bacteria and their stx-negative counterparts have been detected in dairy products. They may convert from the one form to the other by loss or acquisition of Stx phages, potentially confounding food microbiological diagnostic methods based on stx gene detection. Here we investigated the diversity and mobility of Stx phages from human and dairy STEC O26:H11 strains. Evaluation of their rate of in vitro induction, occurring either spontaneously or in the presence of mitomycin C, showed that the Stx2 phages were more inducible overall than Stx1 phages. However, no correlation was found between the Stx phage levels produced and the origin of the strains tested or the phage insertion sites. Morphological analysis by electron microscopy showed that Stx phages from STEC O26:H11 displayed various shapes that were unrelated to Stx1 or Stx2 types. Finally, the levels of sensitivity of stx-negative E. coli O26:H11 to six Stx phages differed among the 17 strains tested and our attempts to convert them into STEC were unsuccessful, indicating that their lysogenization was a rare event.

  11. 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

  12. 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

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

    2015-03-01

    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.

  13. 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.

  14. 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

  15. 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

  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. Tolerance of a Phage Element by Streptococcus pneumoniae Leads to a Fitness Defect during Colonization

    PubMed Central

    DeBardeleben, Hilary K.; Lysenko, Elena S.; Dalia, Ankur B.

    2014-01-01

    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

  19. 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

  20. Puerarin Attenuates Cardiac Hypertrophy Partly Through Increasing Mir-15b/195 Expression and Suppressing Non-Canonical Transforming Growth Factor Beta (Tgfβ) Signal Pathway

    PubMed Central

    Zhang, Xiuzhou; Liu, Yuxiang; Han, Qingliang

    2016-01-01

    Background Previous studies demonstrated that puerarin has therapeutic effects on cardiac hypertrophy. This study aimed to explore whether the effect of puerarin on attenuating cardiac hypertrophy is related to regulation of microRNAs (miRNAs) and the transforming growth factor beta (TGFβ) signal pathway. Material/Methods The therapeutic effect of puerarin was assessed using an angiotensin (Ang) II-induced heart hypertrophy model in mice. The primary cardiomyocytes were used as an in vitro model. MiR-15 family expression was quantified using qRT-PCR analysis. The expression of the genes involved in canonical and non-canonical TGFβ signal pathways was measured using qRT-PCR and Western blot analysis. In vitro cardiac hypertrophic features were assessed by quantifying cardiac hypertrophic genes and measurement of cell surface, protein synthesis, and total protein content. Results Puerarin attenuated cardiac hypertrophy and increased miR-15b and miR-195 expression in the mouse cardiac hypertrophy model and in primary cardiomyocytes. It suppressed both canonical and non-canonical TGFβ signal pathways, partially through miR-15b and miR-195. Puerarin reduced mRNA expression of cardiac hypertrophic genes, reduced cell surface area, and lowered the rate of protein synthesis and the total protein content induced by Ang II. Knockdown of endogenous miR-15b and miR-195 partly abrogated these effects. Knockdown of endogenous p38, but not Smad2/3/4, presented similar effects as miR-15b. Conclusions Puerarin administration enhances miR-15b and miR-195 expression in an Ang II-induced cardiac hypertrophy model, through which it suppresses both canonical and non-canonical TGFβ signal pathways at the same time. However, the effect of puerarin on attenuating cardiac hypertrophy is mainly through the non-canonical TGFβ pathway. PMID:27145790

  1. Galunisertib (LY2157299), a transforming growth factor-β receptor I kinase inhibitor, attenuates acute pancreatitis in rats.

    PubMed

    Liu, X; Yu, M; Chen, Y; Zhang, J

    2016-08-01

    Galunisertib (LY2157299), a selective ATP-mimetic inhibitor of TGF-β receptor I (TGF-βRI), is the only known TGF-β pathway inhibitor. In the present study, we investigated the effect of galunisertib on taurocholate (TAC)-induced acute pancreatitis (AP) in rats, and the role of TGF-β and NF-κB signaling pathways. AP was induced by infusion of TAC into the pancreatic duct of Sprague-Dawley male rats (n=30). The rats (220±50 g) were administered galunisertib intragastrically [75 mg·kg-1·day-1 for 2 days (0 and 24 h)]. Serum IL-1β, IL-6, TNF-α, amylase (AMY), lipase (LIP), and myeloperoxidase (MPO) levels were measured by ELISA. NF-κB activity was detected by electrophoretic mobility shift assay (EMSA); NF-κBp65 and TGF-β1 proteins, as well as TGF-βRI and p-Smad2/3 proteins, were detected by western blot assay. Cell apoptosis was detected by TUNEL assay. H&E staining was used to evaluate the histopathological alterations of the pancreas. Galunisertib treatment attenuated the severity of AP and decreased the pancreatic histological score. In addition, number of apoptotic cells were significantly increased in the galunisertib-treated group (16.38±2.26) than in the AP group (8.14±1.27) and sham-operated group (1.82±0.73; P<0.05 and P<0.01, respectively). Galunisertib decreased the expression levels of TGF-βRI and p-Smad2/3 and inhibited NF-κB activation and p65 translocation compared with the sham-operated group. Furthermore, serum IL-1β, IL-6, TNF-α, AMY and LIP levels and tissue MPO activity were significantly decreased in the galunisertib-treated group. Our data demonstrate that galunisertib attenuates the severity of TAC-induced experimental AP in rats by inducing apoptosis in the pancreas, inhibiting the activation of TGF-β signals and NF-κB as well as the secretion of pro-inflammatory cytokines. PMID:27509307

  2. Galunisertib (LY2157299), a transforming growth factor-β receptor I kinase inhibitor, attenuates acute pancreatitis in rats

    PubMed Central

    Liu, X.; Yu, M.; Chen, Y.; Zhang, J.

    2016-01-01

    Galunisertib (LY2157299), a selective ATP-mimetic inhibitor of TGF-β receptor I (TGF-βRI), is the only known TGF-β pathway inhibitor. In the present study, we investigated the effect of galunisertib on taurocholate (TAC)-induced acute pancreatitis (AP) in rats, and the role of TGF-β and NF-κB signaling pathways. AP was induced by infusion of TAC into the pancreatic duct of Sprague-Dawley male rats (n=30). The rats (220±50 g) were administered galunisertib intragastrically [75 mg·kg-1·day-1 for 2 days (0 and 24 h)]. Serum IL-1β, IL-6, TNF-α, amylase (AMY), lipase (LIP), and myeloperoxidase (MPO) levels were measured by ELISA. NF-κB activity was detected by electrophoretic mobility shift assay (EMSA); NF-κBp65 and TGF-β1 proteins, as well as TGF-βRI and p-Smad2/3 proteins, were detected by western blot assay. Cell apoptosis was detected by TUNEL assay. H&E staining was used to evaluate the histopathological alterations of the pancreas. Galunisertib treatment attenuated the severity of AP and decreased the pancreatic histological score. In addition, number of apoptotic cells were significantly increased in the galunisertib-treated group (16.38±2.26) than in the AP group (8.14±1.27) and sham-operated group (1.82±0.73; P<0.05 and P<0.01, respectively). Galunisertib decreased the expression levels of TGF-βRI and p-Smad2/3 and inhibited NF-κB activation and p65 translocation compared with the sham-operated group. Furthermore, serum IL-1β, IL-6, TNF-α, AMY and LIP levels and tissue MPO activity were significantly decreased in the galunisertib-treated group. Our data demonstrate that galunisertib attenuates the severity of TAC-induced experimental AP in rats by inducing apoptosis in the pancreas, inhibiting the activation of TGF-β signals and NF-κB as well as the secretion of pro-inflammatory cytokines. PMID:27509307

  3. 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

  4. Planetary Ices Attenuation Properties

    NASA Astrophysics Data System (ADS)

    McCarthy, Christine; Castillo-Rogez, Julie C.

    In this chapter, we review the topic of energy dissipation in the context of icy satellites experiencing tidal forcing. We describe the physics of mechanical dissipation, also known as attenuation, in polycrystalline ice and discuss the history of laboratory methods used to measure and understand it. Because many factors - such as microstructure, composition and defect state - can influence rheological behavior, we review what is known about the mechanisms responsible for attenuation in ice and what can be inferred from the properties of rocks, metals and ceramics. Since attenuation measured in the laboratory must be carefully scaled to geologic time and to planetary conditions in order to provide realistic extrapolation, we discuss various mechanical models that have been used, with varying degrees of success, to describe attenuation as a function of forcing frequency and temperature. We review the literature in which these models have been used to describe dissipation in the moons of Jupiter and Saturn. Finally, we address gaps in our present knowledge of planetary ice attenuation and provide suggestions for future inquiry.

  5. A novel thiol compound, N-acetylcysteine amide, attenuates allergic airway disease by regulating activation of NF-kappaB and hypoxia-inducible factor-1alpha.

    PubMed

    Lee, Kyung Sun; Kim, So Ri; Park, Hee Sun; Park, Seoung Ju; Min, Kyung Hoon; Lee, Ka Young; Choe, Yeong Hun; Hong, Sang Hyun; Han, Hyo Jin; Lee, Young Rae; Kim, Jong Suk; Atlas, Daphne; Lee, Yong Chul

    2007-12-31

    Reactive oxygen species (ROS) play an important role in the pathogenesis of airway inflammation and hyperresponsiveness. Recent studies have demonstrated that antioxidants are able to reduce airway inflammation and hyperreactivity in animal models of allergic airway disease. A newly developed antioxidant, small molecular weight thiol compound, N-acetylcysteine amide (AD4) has been shown to increase cellular levels of glutathione and to attenuate oxidative stress related disorders such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. However, the effects of AD4 on allergic airway disease such as asthma are unknown. We used ovalbumin (OVA)-inhaled mice to evaluate the role of AD4 in allergic airway disease. In this study with OVA-inhaled mice, the increased ROS generation, the increased levels of Th2 cytokines and VEGF, the increased vascular permeability, the increased mucus production, and the increased airway resistance in the lungs were significantly reduced by the administration of AD4. We also found that the administration of AD4 decreased the increases of the NF-kappaB and hypoxia-inducible factor-1alpha (HIF-1alpha) levels in nuclear protein extracts of lung tissues after OVA inhalation. These results suggest that AD4 attenuates airway inflammation and hyperresponsiveness by regulating activation of NF-kappaB and HIF-1alpha as well as reducing ROS generation in allergic airway disease. PMID:18160846

  6. A novel thiol compound, N-acetylcysteine amide, attenuates allergic airway disease by regulating activation of NF-kappaB and hypoxia-inducible factor-1alpha.

    PubMed

    Lee, Kyung Sun; Kim, So Ri; Park, Hee Sun; Park, Seoung Ju; Min, Kyung Hoon; Lee, Ka Young; Choe, Yeong Hun; Hong, Sang Hyun; Han, Hyo Jin; Lee, Young Rae; Kim, Jong Suk; Atlas, Daphne; Lee, Yong Chul

    2007-12-31

    Reactive oxygen species (ROS) play an important role in the pathogenesis of airway inflammation and hyperresponsiveness. Recent studies have demonstrated that antioxidants are able to reduce airway inflammation and hyperreactivity in animal models of allergic airway disease. A newly developed antioxidant, small molecular weight thiol compound, N-acetylcysteine amide (AD4) has been shown to increase cellular levels of glutathione and to attenuate oxidative stress related disorders such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. However, the effects of AD4 on allergic airway disease such as asthma are unknown. We used ovalbumin (OVA)-inhaled mice to evaluate the role of AD4 in allergic airway disease. In this study with OVA-inhaled mice, the increased ROS generation, the increased levels of Th2 cytokines and VEGF, the increased vascular permeability, the increased mucus production, and the increased airway resistance in the lungs were significantly reduced by the administration of AD4. We also found that the administration of AD4 decreased the increases of the NF-kappaB and hypoxia-inducible factor-1alpha (HIF-1alpha) levels in nuclear protein extracts of lung tissues after OVA inhalation. These results suggest that AD4 attenuates airway inflammation and hyperresponsiveness by regulating activation of NF-kappaB and HIF-1alpha as well as reducing ROS generation in allergic airway disease.

  7. miR-homoHSV of Singapore grouper iridovirus (SGIV) inhibits expression of the SGIV pro-apoptotic factor LITAF and attenuates cell death.

    PubMed

    Guo, Chuanyu; Yan, Yang; Cui, Huachun; Huang, Xiaohong; Qin, Qiwei

    2013-01-01

    Growing evidence demonstrates that various large DNA viruses could encode microRNAs (miRNAs) that regulate host and viral genes to achieve immune evasion. In this study, we report that miR-homoHSV, an miRNA encoded by Singapore grouper iridovirus (SGIV), can attenuate SGIV-induced cell death. Mechanistically, SGIV miR-homoHSV targets SGIV ORF136R, a viral gene that encodes the pro-apoptotic lipopolysaccharide-induced TNF-α (LITAF)-like factor. miR-homoHSV suppressed exogenous and endogenous SGIV LITAF expression, and thus inhibited SGIV LITAF-induced apoptosis. Meanwhile, miR-homoHSV expression was able to attenuate cell death induced by viral infection, presumably facilitating viral replication through the down-regulation of the pro-apoptotic gene SGIV LITAF. Together, our data suggest miR-homoHSV may serve as a feedback regulator of cell death during viral infection. The findings of this study provide a better understanding of SGIV replication and pathogenesis.

  8. Molecular characterization of a phage-encoded resistance system in Lactococcus lactis.

    PubMed

    McGrath, S; Seegers, J F; Fitzgerald, G F; van Sinderen, D

    1999-05-01

    A specific fragment of the genome of Tuc2009, a temperate lactococcal bacteriophage, was shown to contain several open reading frames, whose deduced protein products exhibited similarities to proteins known to be involved in DNA replication and modification. In this way, a putative single-stranded binding protein, replisome organizer protein, topoisomerase I, and a methylase were identified. When the genetic information coding for the putative replisome organizer protein of Tuc2009, Rep2009, was supplied on a high-copy-number plasmid vector, it was shown to confer a phage-encoded resistance (Per) phenotype on its lactococcal host UC509.9. The presence of this recombinant plasmid was shown to cause a marked reduction in Tuc2009 DNA replication, suggesting that the observed phage resistance was due to titration of a factor, or factors, required for Tuc2009 DNA replication. Further experiments delineated the phage resistance-conferring region to a 160-bp fragment rich in direct repeats. Gel retardation experiments, which indicated a protein-DNA interaction between this 160-bp fragment and the Rep2009 protein, were performed. UC509.9 strains harboring plasmids with randomly mutated versions of this fragment were shown to display a variable phage resistance phenotype, depending on the position of the mutations.

  9. 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.

  10. Convergent evolution of pathogenicity islands in helper cos phage interference.

    PubMed

    Carpena, Nuria; Manning, Keith A; Dokland, Terje; Marina, Alberto; Penadés, José R

    2016-11-01

    Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful (pac) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpmA and cpmB genes. Another SaPI subfamily is induced and packaged by cos-type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm, which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution.This article is part of the themed issue 'The new bacteriology'.

  11. 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

  12. Convergent evolution of pathogenicity islands in helper cos phage interference

    PubMed Central

    Manning, Keith A.; Dokland, Terje; Marina, Alberto

    2016-01-01

    Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful (pac) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpmA and cpmB genes. Another SaPI subfamily is induced and packaged by cos-type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm, which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution. This article is part of the themed issue ‘The new bacteriology’. PMID:27672154

  13. Convergent evolution of pathogenicity islands in helper cos phage interference.

    PubMed

    Carpena, Nuria; Manning, Keith A; Dokland, Terje; Marina, Alberto; Penadés, José R

    2016-11-01

    Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful (pac) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpmA and cpmB genes. Another SaPI subfamily is induced and packaged by cos-type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm, which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution.This article is part of the themed issue 'The new bacteriology'. PMID:27672154

  14. 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

  15. 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.

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

    PubMed Central

    Holmfeldt, Karin; Solonenko, Natalie; Shah, Manesh; Corrier, Kristen; 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 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

  17. Reduced-calorie avocado paste attenuates metabolic factors associated with a hypercholesterolemic-high fructose diet in rats.

    PubMed

    Pahua-Ramos, María Elena; Garduño-Siciliano, Leticia; Dorantes-Alvarez, Lidia; Chamorro-Cevallos, German; Herrera-Martínez, Julieta; Osorio-Esquivel, Obed; Ortiz-Moreno, Alicia

    2014-03-01

    The objective of this study was to evaluate the effect of reduced-calorie avocado paste on lipid serum profile, insulin sensitivity, and hepatic steatosis in rats fed a hypercholesterolemic-high fructose diet. Thirty five male Wistar rats were randomly separated in five groups: Control group (ground commercial diet); hypercholesterolemic diet plus 60% fructose solution (HHF group); hypercholesterolemic diet plus 60% fructose solution supplemented with avocado pulp (HHF+A group); hypercholesterolemic diet plus 60% fructose solution supplemented with reduced-calorie avocado paste (HHF+P group); and hypercholesterolemic diet plus 60% fructose solution supplemented with a reduced-calorie avocado paste plus fiber (HHF+FP group). The A, P, and FP were supplemented at 2 g/kg/d. The study was carried out for seven weeks. Rats belonging to the HHF group exhibited significantly (P ≤ 0.05) higher total cholesterol, triglycerides, and insulin levels in serum as well as lower insulin sensitivity than the control group. Supplementation with reduced-calorie avocado paste showed a significant (P ≤ 0.05) decrease in total cholesterol (43.1%), low-density lipoprotein (45.4%), and triglycerides (32.8%) in plasma as well as elevated insulin sensitivity compared to the HHF group. Additionally, the liver enzymes alanine aminotransferase and aspartate aminotransferase decreased significantly in the HHF-P group (39.8 and 35.1%, respectively). These results are likely due to biocompounds present in the reduced-calorie avocado paste, such as polyphenols, carotenoids, chlorophylls, and dietary fibre, which are capable of reducing oxidative stress. Therefore, reduced-calorie avocado paste attenuates the effects of a hypercholesterolemic-high fructose diet in rats.

  18. Tumor necrosis factorattenuates starvation-induced apoptosis through upregulation of ferritin heavy chain in hepatocellular carcinoma cells

    PubMed Central

    2013-01-01

    Background Tumor microenviroment is characteristic of inflammation, ischemia and starvation of nutrient. TNF-α, which is an extraordinarily pleiotropic cytokine, could be an endogenous tumor promoter in some tumor types. The basic objective of this study was to investigate the effects of TNF-α on the cell viability and apoptosis of hepatocellular carcinoma cells under serum starvation, and to identify the molecular mechanisms involved. Methods For this purpose, five different concentrations of TNF-α and two different serum settings (serum-cultured and serum-deprived) were used to investigate the effects of TNF-α on the cell viability and apoptosis of Hep3B and SMMC-7721 cells. Results TNF-α (10 ng/ml) attenuated serum starvation-induced apoptosis of hepatocellular carcinoma cells, and autophagy conferred this process. BAY11-7082, a specific inhibitor of NF-κB, reversed the suppression of serum starvation-induced apoptosis by TNF-α. Moreover, TNF-α-induced NF-κB transactivation was suppressed by autophagy inhibitor 3-MA. In addition, TNF-α up-regulated Ferritin heavy chain (FHC) transiently by NF-κB activation and FHC levels were correlated with the TNF-α-induced protection against serum starvation-mediated apoptosis of hepatocellular carcinoma cells. Furthermore, FHC-mediated inhibition of apoptosis depended on suppressing ROS accumulation. Conclusions Our findings suggested that autophagy conferred the TNF-α protection against serum starvation-mediated apoptosis of hepatocellular carcinoma cells, the mechanism involved with the activation of the TNF-α/ NF-κB /FHC signaling pathway. PMID:24066693

  19. 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

  20. Progress in lactic acid bacterial phage research

    PubMed Central

    2014-01-01

    Research on lactic acid bacteria (LAB) has advanced significantly over the past number of decades and these developments have been driven by the parallel advances in technologies such as genomics, bioinformatics, protein expression systems and structural biology, combined with the ever increasing commercial relevance of this group of microorganisms. Some of the more significant and impressive outputs have been in the domain of bacteriophage-host interactions which provides a prime example of the cutting-edge model systems represented by LAB research. Here, we present a retrospective overview of the key advances in LAB phage research including phage-host interactions and co-evolution. We describe how in many instances this knowledge can be pivotal in creating real improvements in the application of LAB cultures in commercial practice. PMID:25185514

  1. Large scale production of phage antibody libraries using a bioreactor.

    PubMed

    Ferrara, Fortunato; Kim, Chang-Yub; Naranjo, Leslie A; Bradbury, Andrew R M

    2015-01-01

    One of the limitations of the use of phage antibody libraries in high throughput selections is the production of sufficient phage antibody library at the appropriate quality. Here, we successfully adapt a bioreactor-based protocol for the production of phage peptide libraries to the production of phage antibody libraries. The titers obtained in the stirred-tank bioreactor are 4 to 5 times higher than in a standard shake flask procedure, and the quality of the phage antibody library produced is indistinguishable to that produced using standard procedures as assessed by Western blotting and functional selections. Availability of this protocol will facilitate the use of phage antibody libraries in high-throughput scale selections.

  2. A century of the phage: past, present and future.

    PubMed

    Salmond, George P C; Fineran, Peter C

    2015-12-01

    Viruses that infect bacteria (bacteriophages; also known as phages) were discovered 100 years ago. Since then, phage research has transformed fundamental and translational biosciences. For example, phages were crucial in establishing the central dogma of molecular biology - information is sequentially passed from DNA to RNA to proteins - and they have been shown to have major roles in ecosystems, and help drive bacterial evolution and virulence. Furthermore, phage research has provided many techniques and reagents that underpin modern biology - from sequencing and genome engineering to the recent discovery and exploitation of CRISPR-Cas phage resistance systems. In this Timeline, we discuss a century of phage research and its impact on basic and applied biology.

  3. Trivalent Cation Induced Bundle Formation of Filamentous fd Phages.

    PubMed

    Korkmaz Zirpel, Nuriye; Park, Eun Jin

    2015-09-01

    Bacteriophages are filamentous polyelectrolyte viral rods infecting only bacteria. In this study, we investigate the bundle formation of fd phages with trivalent cations having different ionic radii (Al(3+) , La(3+) and Y(3+) ) at various phage and counterion concentrations, and at varying bundling times. Aggregated phage bundles were detected at relatively low trivalent counterion concentrations (1 mM). Although 10 mM and 100 mM Y(3+) and La(3+) treatments formed larger and more intertwined phage bundles, Al(3+) and Fe(3+) treatments lead to the formation of networking filaments. Energy dispersive X-ray spectroscopy (EDX) analyses confirmed the presence of C, N and O peaks on densely packed phage bundles. Immunofluorescence labelling and ELISA analyses with anti-p8 antibodies showed the presence of phage filaments after bundling.

  4. Assessment of factors influencing PM mass concentration measured by gravimetric & beta attenuation techniques at a suburban site

    NASA Astrophysics Data System (ADS)

    Triantafyllou, E.; Diapouli, E.; Tsilibari, E. M.; Adamopoulos, A. D.; Biskos, G.; Eleftheriadis, K.

    2016-04-01

    Near real-time atmospheric particulate matter (PM) monitors are extensively used in air quality networks given their ability to provide continuous measurements with minimal attention by the operator. Their principle of operation is based on measurement of a physical parameter that is quantitatively linked to the PM mass concentration. Significant discrepancies between these measurements and those obtained by the reference gravimetric method, conducted in regions with diverse climatic conditions, have been reported in the literature. In this study we compare systematic PM2.5 and PM10 gravimetric (GM) and beta attenuation (BA) measurements performed at a suburban site in Athens, Greece, over a period of 4 years (2009-2012). In general, BA and GM datasets exhibited similar temporal variation for both PM size fractions. An overestimation of the ΒΑ measurements, which was ∼30% for the PM2.5 and ∼10% for the PM10 data, was observed. Good linear correlations between GM and BA data were observed, with estimated Pearson coefficients being 0.79 for the PM2.5 and 0.85 for the PM10 measurements. The respective fitted equations through the entire dataset were BA = 0.71 GM + 6.2, and BA = 0.77 G M + 4.1. Better correlation between GM and BA measurements was observed during the cold rather than the warm period. Discrepancies between BA and GM PM2.5 measurements increased with increasing available water vapor, suggesting that the aerosol bound water has a strong effect on the measurements. The effect of filter material used for GM measurements (i.e., quartz, glass fiber, or Teflon) was also examined for the PM2.5 dataset. Best correlation between BA and GM data was observed when glass fiber, which is incidentally the material of the BA filter tape, was used in the GM measurements. When the BA to GM relationship was examined by further categorizing the data by the season (i.e., cold and warm period) for different filter types, the relationships that were fitted to the data

  5. Lysogenic conversion and phage resistance development in phage exposed Escherichia coli biofilms.

    PubMed

    Moons, Pieter; Faster, David; Aertsen, Abram

    2013-01-01

    In this study, three-day old mature biofilms of Escherichia coli were exposed once to either a temperate Shiga-toxin encoding phage (H-19B) or an obligatory lytic phage (T7), after which further dynamics in the biofilm were monitored. As such, it was found that a single dose of H-19B could rapidly lead to a near complete lysogenization of the biofilm, with a subsequent continuous release of infectious H-19B particles. On the other hand, a single dose of T7 rapidly led to resistance development in the biofilm population. Together, our data indicates a profound impact of phages on the dynamics within structured bacterial populations. PMID:23344561

  6. Loss of ADAM17-Mediated Tumor Necrosis Factor Alpha Signaling in Intestinal Cells Attenuates Mucosal Atrophy in a Mouse Model of Parenteral Nutrition.

    PubMed

    Feng, Yongjia; Tsai, Yu-Hwai; Xiao, Weidong; Ralls, Matthew W; Stoeck, Alex; Wilson, Carole L; Raines, Elaine W; Teitelbaum, Daniel H; Dempsey, Peter J

    2015-11-01

    Total parenteral nutrition (TPN) is commonly used clinically to sustain patients; however, TPN is associated with profound mucosal atrophy, which may adversely affect clinical outcomes. Using a mouse TPN model, removing enteral nutrition leads to decreased crypt proliferation, increased intestinal epithelial cell (IEC) apoptosis and increased mucosal tumor necrosis factor alpha (TNF-α) expression that ultimately produces mucosal atrophy. Upregulation of TNF-α signaling plays a central role in mediating TPN-induced mucosal atrophy without intact epidermal growth factor receptor (EGFR) signaling. Currently, the mechanism and the tissue-specific contributions of TNF-α signaling to TPN-induced mucosal atrophy remain unclear. ADAM17 is an ectodomain sheddase that can modulate the signaling activity of several cytokine/growth factor receptor families, including the TNF-α/TNF receptor and ErbB ligand/EGFR pathways. Using TPN-treated IEC-specific ADAM17-deficient mice, the present study demonstrates that a loss of soluble TNF-α signaling from IECs attenuates TPN-induced mucosal atrophy. Importantly, this response remains dependent on the maintenance of functional EGFR signaling in IECs. TNF-α blockade in wild-type mice receiving TPN confirmed that soluble TNF-α signaling is responsible for downregulation of EGFR signaling in IECs. These results demonstrate that ADAM17-mediated TNF-α signaling from IECs has a significant role in the development of the proinflammatory state and mucosal atrophy observed in TPN-treated mice.

  7. Quetiapine Attenuates Glial Activation and Proinflammatory Cytokines in APP/PS1 Transgenic Mice via Inhibition of Nuclear Factor-κB Pathway

    PubMed Central

    Zhu, Shenghua; Shi, Ruoyang; Li, Victor; Wang, Junhui; Zhang, Ruiguo; Tempier, Adrien; He, Jue; Kong, Jiming; Wang, Jun-Feng

    2015-01-01

    Background: In Alzheimer’s disease, growing evidence has shown that uncontrolled glial activation and neuroinflammation may contribute independently to neurodegeneration. Antiinflammatory strategies might provide benefits for this devastating disease. The aims of the present study are to address the issue of whether glial activation and proinflammatory cytokine increases could be modulated by quetiapine in vivo and in vitro and to explore the underlying mechanism. Methods: Four-month–old amyloid precursor protein (APP) and presenilin 1 (PS1) transgenic and nontransgenic mice were treated with quetiapine (5mg/kg/d) in drinking water for 8 months. Animal behaviors, total Aβ levels, and glial activation were evaluated by behavioral tests, enzyme-linked immunosorbent assay, immunohistochemistry, and Western blot accordingly. Inflammatory cytokines and the nuclear factor kappa B pathway were analyzed in vivo and in vitro. Results: Quetiapine improves behavioral performance, marginally affects total Aβ40 and Aβ42 levels, attenuates glial activation, and reduces proinflammatory cytokines in APP/PS1 mice. Quetiapine suppresses Aβ1-42-induced activation of primary microglia by decresing proinflammatory cytokines. Quetiapine inhibits the activation of nuclear factor kappa B p65 pathway in both transgenic mice and primary microglia stimulated by Aβ1–42. Conclusions: The antiinflammatory effects of quetiapine in Alzheimer’s disease may be involved in the nuclear factor kappa B pathway. Quetiapine may be an efficacious and promising treatment for Alzheimer’s disease targeting on neuroinflammation. PMID:25618401

  8. Loss of ADAM17-Mediated Tumor Necrosis Factor Alpha Signaling in Intestinal Cells Attenuates Mucosal Atrophy in a Mouse Model of Parenteral Nutrition

    PubMed Central

    Feng, Yongjia; Tsai, Yu-Hwai; Xiao, Weidong; Ralls, Matthew W.; Stoeck, Alex; Wilson, Carole L.; Raines, Elaine W.

    2015-01-01

    Total parenteral nutrition (TPN) is commonly used clinically to sustain patients; however, TPN is associated with profound mucosal atrophy, which may adversely affect clinical outcomes. Using a mouse TPN model, removing enteral nutrition leads to decreased crypt proliferation, increased intestinal epithelial cell (IEC) apoptosis and increased mucosal tumor necrosis factor alpha (TNF-α) expression that ultimately produces mucosal atrophy. Upregulation of TNF-α signaling plays a central role in mediating TPN-induced mucosal atrophy without intact epidermal growth factor receptor (EGFR) signaling. Currently, the mechanism and the tissue-specific contributions of TNF-α signaling to TPN-induced mucosal atrophy remain unclear. ADAM17 is an ectodomain sheddase that can modulate the signaling activity of several cytokine/growth factor receptor families, including the TNF-α/TNF receptor and ErbB ligand/EGFR pathways. Using TPN-treated IEC-specific ADAM17-deficient mice, the present study demonstrates that a loss of soluble TNF-α signaling from IECs attenuates TPN-induced mucosal atrophy. Importantly, this response remains dependent on the maintenance of functional EGFR signaling in IECs. TNF-α blockade in wild-type mice receiving TPN confirmed that soluble TNF-α signaling is responsible for downregulation of EGFR signaling in IECs. These results demonstrate that ADAM17-mediated TNF-α signaling from IECs has a significant role in the development of the proinflammatory state and mucosal atrophy observed in TPN-treated mice. PMID:26283731

  9. Therapeutic effect of Pseudomonas aeruginosa phage YH30 on mink hemorrhagic pneumonia.

    PubMed

    Gu, Jingmin; Li, Xinwei; Yang, Mei; Du, Chongtao; Cui, Ziyin; Gong, Pengjuan; Xia, Feifei; Song, Jun; Zhang, Lei; Li, Juecheng; Yu, Chuang; Sun, Changjiang; Feng, Xin; Lei, Liancheng; Han, Wenyu

    2016-07-15

    Hemorrhagic pneumonia caused by Pseudomonas aeruginosa remains one of the most costly infectious diseases among farmed mink and commonly leads to large economic losses during mink production. The objective of this study was to investigate the potential of using phages as a therapy against hemorrhagic pneumonia in mink. A broad-host-range phage from the Podoviridae family, YH30, was isolated using the mink-originating P. aeruginosa (serotype G) D7 strain as a host. The genome of YH30 was 72,192bp (54.92% G+C), contained 86 open reading frames and lacked regions encoding known virulence factors, integration-related proteins or antibiotic resistance determinants. These characteristics make YH30 eligible for use in phage therapy. The results of a curative treatment experiment demonstrated that a single intranasal administration of YH30 was sufficient to cure hemorrhagic pneumonia in mink. The mean colony count of P. aeruginosa in the blood and lung of YH30-protected mink was less than 10(3) CFU/mL (g) within 24h of bacterial challenge and ultimately became undetectable, whereas that in unprotected mink reached more than 10(8) CFU/mL (g). Additionally, YH30 dramatically improved the pathological manifestations of lung injury in mink with hemorrhagic pneumonia. Our work demonstrates the potential of phages to treat P. aeruginosa-caused hemorrhagic pneumonia in mink. PMID:27283850

  10. Identification of Novel Single Chain Fragment Variable Antibodies Against TNF-α Using Phage Display Technology

    PubMed Central

    Alizadeh, Ali Akbar; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

    2015-01-01

    Purpose: Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological and pathological pathways. Because of central role of TNF-α in pathogenesis of inflammatory diseases, in the current study, we aimed to identify novel scFv antibodies against TNF-α using phage display technology. Methods: Using libraries composed of phagemid displaying scFv antibodies, four rounds of biopanning against TNF-α were carried out, which led to identification of scFvs capable of binding to TNF-α. The scFv antibody with appropriate binding affinity towards TNF-α, was amplified and used in ELISA experiment. Results: Titration of phage achieved from different rounds of biopanning showed an enrichment of specific anti-TNF-α phages during biopanning process. Using ELISA experiment, a binding constant (Kd) of 1.11 ± 0.32 nM was determined for the phage displaying J48 scFv antibody. Conclusion: The findings in the current work revealed that the identified novel scFv antibody displayed at the N-terminal of minor coat proteins of phagemid binds TNF-α with suitable affinity. However, the soluble form of the antibody is needed to be produced and evaluated in more details regarding its binding properties to TNF-α. PMID:26793613

  11. Satellite phage TLCφ enables toxigenic conversion by CTX phage through dif site alteration.

    PubMed

    Hassan, Faizule; Kamruzzaman, M; Mekalanos, John J; Faruque, Shah M

    2010-10-21

    Bacterial chromosomes often carry integrated genetic elements (for example plasmids, transposons, prophages and islands) whose precise function and contribution to the evolutionary fitness of the host bacterium are unknown. The CTXφ prophage, which encodes cholera toxin in Vibrio cholerae, is known to be adjacent to a chromosomally integrated element of unknown function termed the toxin-linked cryptic (TLC). Here we report the characterization of a TLC-related element that corresponds to the genome of a satellite filamentous phage (TLC-Knφ1), which uses the morphogenesis genes of another filamentous phage (fs2φ) to form infectious TLC-Knφ1 phage particles. The TLC-Knφ1 phage genome carries a sequence similar to the dif recombination sequence, which functions in chromosome dimer resolution using XerC and XerD recombinases. The dif sequence is also exploited by lysogenic filamentous phages (for example CTXφ) for chromosomal integration of their genomes. Bacterial cells defective in the dimer resolution often show an aberrant filamentous cell morphology. We found that acquisition and chromosomal integration of the TLC-Knφ1 genome restored a perfect dif site and normal morphology to V. cholerae wild-type and mutant strains with dif(-) filamentation phenotypes. Furthermore, lysogeny of a dif(-) non-toxigenic V. cholerae with TLC-Knφ1 promoted its subsequent toxigenic conversion through integration of CTXφ into the restored dif site. These results reveal a remarkable level of cooperative interactions between multiple filamentous phages in the emergence of the bacterial pathogen that causes cholera. PMID:20944629

  12. Learning from Bacteriophages - Advantages and Limitations of Phage and Phage-Encoded Protein Applications

    PubMed Central

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grażyna; Maciejewska, Barbara; Delattre, Anne-Sophie; Lavigne, Rob

    2012-01-01

    The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application. PMID:23305359

  13. Bacteriophages and phage-derived proteins--application approaches.

    PubMed

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grazyna; Maciejewska, Barbara

    2015-01-01

    Currently, the bacterial resistance, especially to most commonly used antibiotics has proved to be a severe therapeutic problem. Nosocomial and community-acquired infections are usually caused by multidrug resistant strains. Therefore, we are forced to develop an alternative or supportive treatment for successful cure of life-threatening infections. The idea of using natural bacterial pathogens such as bacteriophages is already well known. Many papers have been published proving the high antibacterial efficacy of lytic phages tested in animal models as well as in the clinic. Researchers have also investigated the application of non-lytic phages and temperate phages, with promising results. Moreover, the development of molecular biology and novel generation methods of sequencing has opened up new possibilities in the design of engineered phages and recombinant phage-derived proteins. Encouraging performances were noted especially for phage enzymes involved in the first step of viral infection responsible for bacterial envelope degradation, named depolymerases. There are at least five major groups of such enzymes - peptidoglycan hydrolases, endosialidases, endorhamnosidases, alginate lyases and hyaluronate lyases - that have application potential. There is also much interest in proteins encoded by lysis cassette genes (holins, endolysins, spanins) responsible for progeny release during the phage lytic cycle. In this review, we discuss several issues of phage and phage-derived protein application approaches in therapy, diagnostics and biotechnology in general. PMID:25666799

  14. Isolation and Characterization of Phages Infecting Bacillus subtilis

    PubMed Central

    Krasowska, Anna; Biegalska, Anna; Augustyniak, Daria; Łoś, Marcin; Richert, Malwina; Łukaszewicz, Marcin

    2015-01-01

    Bacteriophages have been suggested as an alternative approach to reduce the amount of pathogens in various applications. Bacteriophages of various specificity and virulence were isolated as a means of controlling food-borne pathogens. We studied the interaction of bacteriophages with Bacillus species, which are very often persistent in industrial applications such as food production due to their antibiotic resistance and spore formation. A comparative study using electron microscopy, PFGE, and SDS-PAGE as well as determination of host range, pH and temperature resistance, adsorption rate, latent time, and phage burst size was performed on three phages of the Myoviridae family and one phage of the Siphoviridae family which infected Bacillus subtilis strains. The phages are morphologically different and characterized by icosahedral heads and contractile (SIOΦ, SUBω, and SPOσ phages) or noncontractile (ARπ phage) tails. The genomes of SIOΦ and SUBω are composed of 154 kb. The capsid of SIOΦ is composed of four proteins. Bacteriophages SPOσ and ARπ have genome sizes of 25 kbp and 40 kbp, respectively. Both phages as well as SUBω phage have 14 proteins in their capsids. Phages SIOΦ and SPOσ are resistant to high temperatures and to the acid (4.0) and alkaline (9.0 and 10.0) pH. PMID:26273592

  15. Bacteriophages and Phage-Derived Proteins – Application Approaches

    PubMed Central

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grazyna; Maciejewska, Barbara

    2015-01-01

    Currently, the bacterial resistance, especially to most commonly used antibiotics has proved to be a severe therapeutic problem. Nosocomial and community-acquired infections are usually caused by multidrug resistant strains. Therefore, we are forced to develop an alternative or supportive treatment for successful cure of life-threatening infections. The idea of using natural bacterial pathogens such as bacteriophages is already well known. Many papers have been published proving the high antibacterial efficacy of lytic phages tested in animal models as well as in the clinic. Researchers have also investigated the application of non-lytic phages and temperate phages, with promising results. Moreover, the development of molecular biology and novel generation methods of sequencing has opened up new possibilities in the design of engineered phages and recombinant phage-derived proteins. Encouraging performances were noted especially for phage enzymes involved in the first step of viral infection responsible for bacterial envelope degradation, named depolymerases. There are at least five major groups of such enzymes – peptidoglycan hydrolases, endosialidases, endorhamnosidases, alginate lyases and hyaluronate lyases – that have application potential. There is also much interest in proteins encoded by lysis cassette genes (holins, endolysins, spanins) responsible for progeny release during the phage lytic cycle. In this review, we discuss several issues of phage and phage-derived protein application approaches in therapy, diagnostics and biotechnology in general. PMID:25666799

  16. Quality and safety requirements for sustainable phage therapy products.

    PubMed

    Pirnay, Jean-Paul; Blasdel, Bob G; Bretaudeau, Laurent; Buckling, Angus; Chanishvili, Nina; Clark, Jason R; Corte-Real, Sofia; Debarbieux, Laurent; Dublanchet, Alain; De Vos, Daniel; Gabard, Jérôme; Garcia, Miguel; Goderdzishvili, Marina; Górski, Andrzej; Hardcastle, John; Huys, Isabelle; Kutter, Elizabeth; Lavigne, Rob; Merabishvili, Maia; Olchawa, Ewa; Parikka, Kaarle J; Patey, Olivier; Pouilot, Flavie; Resch, Gregory; Rohde, Christine; Scheres, Jacques; Skurnik, Mikael; Vaneechoutte, Mario; Van Parys, Luc; Verbeken, Gilbert; Zizi, Martin; Van den Eede, Guy

    2015-07-01

    The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Since time immemorial phages control bacterial populations on Earth. Potent lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. In addition, phages have the capacity to rapidly overcome bacterial resistances, which will inevitably emerge. To maximally exploit these advantage phages have over conventional drugs such as antibiotics, it is important that sustainable phage products are not submitted to the conventional long medicinal product development and licensing pathway. There is a need for an adapted framework, including realistic production and quality and safety requirements, that allows a timely supplying of phage therapy products for 'personalized therapy' or for public health or medical emergencies. This paper enumerates all phage therapy product related quality and safety risks known to the authors, as well as the tests that can be performed to minimize these risks, only to the extent needed to protect the patients and to allow and advance responsible phage therapy and research.

  17. Ligand-directed profiling of organelles with internalizing phage libraries

    PubMed Central

    Dobroff, Andrey S.; Rangel, Roberto; Guzman-Roja, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Bologa, Cristian G.; Oprea, Tudor I.; Brinker, C. Jeffrey; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    Phage display is a resourceful tool to, in an unbiased manner, discover and characterize functional protein-protein interactions, to create vaccines, and to engineer peptides, antibodies, and other proteins as targeted diagnostic and/or therapeutic agents. Recently, our group has developed a new class of internalizing phage (iPhage) for ligand-directed targeting of organelles and/or to identify molecular pathways within live cells. This unique technology is suitable for applications ranging from fundamental cell biology to drug development. Here we describe the method for generating and screening the iPhage display system, and explain how to select and validate candidate internalizing homing peptide. PMID:25640897

  18. Distribution of Salmonella enteritidis phage types in Canada.

    PubMed Central

    Khakhria, R.; Duck, D.; Lior, H.

    1991-01-01

    The distribution of Salmonella enteritidis phage types in Canada is described; 606 of 674 strains examined were of human origin. Typable strains of all sources, constituted 99.6% (671/674) of all strains examined, and were representative of 15 different phage types. Five phage types (8, 13, 4, 13a and 1) accounted for 92.4% of the total. Phage type 8 consistently showed the highest incidence in human (69.96%) and non-human (72.05%) sources and appeared to be the most common in North America. Phage type 4, the most prevalent in the UK, is infrequent in Canada (38/674). The distribution of phage types showed regional variation among infrequent phage types, whereas the common type, 8, was observed in different frequencies in all provinces. Examination of 29 outbreaks of S. enteritidis representing 254 isolates for humans revealed 5 different phage types, the highest number of outbreaks (11) were type 8. A study of these outbreaks and the animal-host-associations of the common phage types, 8 and 13, indicated that contaminated poultry appeared to be the most common source of human infection in Canada. PMID:1993451

  19. Identification of Chondrocyte-Binding Peptides by Phage Display

    PubMed Central

    Cheung, Crystal S.F.; Lui, Julian C.; Baron, Jeffrey

    2016-01-01

    As an initial step toward targeting cartilage tissue for potential therapeutic applications, we sought cartilage-binding peptides using phage display, a powerful technology for selection of peptides that bind to molecules of interest. A library of phage displaying random 12-amino acid peptides was iteratively incubated with cultured chondrocytes to select phage that bind cartilage. The resulting phage clones demonstrated increased affinity to chondrocytes by ELISA, when compared to a wild-type, insertless phage. Furthermore, the selected phage showed little preferential binding to other cell types, including primary skin fibroblast, myocyte and hepatocyte cultures, suggesting a tissue-specific interaction. Immunohistochemical staining revealed that the selected phage bound chondrocytes themselves and the surrounding extracellular matrix. FITC-tagged peptides were synthesized based on the sequence of cartilage-binding phage clones. These peptides, but not a random peptide, bound cultured chondrocytes, and extracelluar matrix. In conclusion, using phage display, we identified peptide sequences that specifically target chondrocytes. We anticipate that such peptides may be coupled to therapeutic molecules to provide targeted treatment for cartilage disorders. PMID:23440926

  20. Complete Genome Sequences of Four Novel Escherichia coli Bacteriophages Belonging to New Phage Groups

    PubMed Central

    Kot, Witold

    2015-01-01

    Here, we describe the sequencing and genome annotations of a set of four Escherichia coli bacteriophages (phages) belonging to newly discovered groups previously consisting of only a single phage and thus expand our knowledge of these phage groups. PMID:26184932

  1. Cucurbitacin I Attenuates Cardiomyocyte Hypertrophy via Inhibition of Connective Tissue Growth Factor (CCN2) and TGF- β/Smads Signalings.

    PubMed

    Jeong, Moon Hee; Kim, Shang-Jin; Kang, Hara; Park, Kye Won; Park, Woo Jin; Yang, Seung Yul; Yang, Dong Kwon

    2015-01-01

    Cucurbitacin I is a naturally occurring triterpenoid derived from Cucurbitaceae family plants that exhibits a number of potentially useful pharmacological and biological activities. However, the therapeutic impact of cucurbitacin I on the heart has not heretofore been reported. To evaluate the functional role of cucurbitacin I in an in vitro model of cardiac hypertrophy, phenylephrine (PE)-stimulated cardiomyocytes were treated with a sub-cytotoxic concentration of the compound, and the effects on cell size and mRNA expression levels of ANF and β-MHC were investigated. Consequently, PE-induced cell enlargement and upregulation of ANF and β-MHC were significantly suppressed by pretreatment of the cardiomyocytes with cucurbitacin I. Notably, cucurbitacin I also impaired connective tissue growth factor (CTGF) and MAPK signaling, pro-hypertrophic factors, as well as TGF-β/Smad signaling, the important contributing factors to fibrosis. The protective impact of cucurbitacin I was significantly blunted in CTGF-silenced or TGF-β1-silenced hypertrophic cardiomyocytes, indicating that the compound exerts its beneficial actions through CTGF. Taken together, these findings signify that cucurbitacin I protects the heart against cardiac hypertrophy via inhibition of CTGF/MAPK, and TGF- β/Smad-facilitated events. Accordingly, the present study provides new insights into the defensive capacity of cucurbitacin I against cardiac hypertrophy, and further suggesting cucurbitacin I's utility as a novel therapeutic agent for the management of heart diseases. PMID:26296085

  2. BMP4 Is a Peripherally-Derived Factor for Motor Neurons and Attenuates Glutamate-Induced Excitotoxicity In Vitro

    PubMed Central

    Chou, Hui-Ju; Lai, Dar-Ming; Huang, Cheng-Wen; McLennan, Ian S.; Wang, Horng-Dar; Wang, Pei-Yu

    2013-01-01

    Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-β) superfamily, have been shown to play important roles in the nervous system, including neuronal survival and synaptogenesis. However, the physiological functions of BMP signaling in the mammalian neuromuscular system are not well understood. In this study, we found that proteins of the type II bone morphogenetic receptors (BMPRII) were detected at the neuromuscular junction (NMJ), and one of its ligands, BMP4, was expressed by Schwann cells and skeletal muscle fibers. In double-ligated nerves, BMP4 proteins accumulated at the proximal and distal portions of the axons, suggesting that Schwann cell- and muscle fiber-derived BMP4 proteins were anterogradely and retrogradely transported by motor neurons. Furthermore, BMP4 mRNA was down-regulated in nerves but up-regulated in skeletal muscles following nerve ligation. The motor neuron-muscle interactions were also demonstrated using differentiated C2C12 muscle cells and NG108-15 neurons in vitro. BMP4 mRNA and immunoreactivity were significantly up-regulated in differentiated C2C12 muscle cells when the motor neuron-derived factor, agrin, was present in the culture. Peripherally-derived BMP4, on the other hand, promotes embryonic motor neuron survival and protects NG108-15 neurons from glutamate-induced excitotoxicity. Together, these data suggest that BMP4 is a peripherally-derived factor that may regulate the survival of motor neurons. PMID:23472198

  3. Silibinin attenuates amyloid beta(25-35) peptide-induced memory impairments: implication of inducible nitric-oxide synthase and tumor necrosis factor-alpha in mice.

    PubMed

    Lu, P; Mamiya, T; Lu, L L; Mouri, A; Niwa, M; Hiramatsu, M; Zou, L B; Nagai, T; Ikejima, T; Nabeshima, T

    2009-10-01

    In Alzheimer's disease (AD), the deposition of amyloid peptides is invariably associated with oxidative stress and inflammatory responses. Silibinin (silybin), a flavonoid derived from the herb milk thistle, has potent anti-inflammatory and antioxidant activities. However, it remains unclear whether silibinin improves amyloid beta (Abeta) peptide-induced neurotoxicity. In this study, we examined the effect of silibinin on the fear-conditioning memory deficits, inflammatory response, and oxidative stress induced by the intracerebroventricular injection of Abeta peptide(25-35) (Abeta(25-35)) in mice. Mice were treated with silibinin (2, 20, and 200 mg/kg p.o., once a day for 8 days) from the day of the Abeta(25-35) injection (day 0). Memory function was evaluated in cued and contextual fear-conditioning tests (day 6). Nitrotyrosine levels in the hippocampus and amygdala were examined (day 8). The mRNA expression of inducible nitric-oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) in the hippocampus and amygdala was measured 2 h after the Abeta(25-35) injection. We found that silibinin significantly attenuated memory deficits caused by Abeta(25-35) in the cued and contextual fear-conditioning test. Silibinin significantly inhibited the increase in nitrotyrosine levels in the hippocampus and amygdala induced by Abeta(25-35). Nitrotyrosine levels in these regions were negatively correlated with memory performance. Moreover, real-time RT-PCR revealed that silibinin inhibited the overexpression of iNOS and TNF-alpha mRNA in the hippocampus and amygdala induced by Abeta(25-35). These findings suggest that silibinin (i) attenuates memory impairment through amelioration of oxidative stress and inflammatory response induced by Abeta(25-35) and (ii) may be a potential candidate for an AD medication. PMID:19638571

  4. Antagonism of corticotrophin-releasing factor type 1 receptors attenuates caloric intake of free feeding subordinate female rhesus monkeys in a rich dietary environment.

    PubMed

    Moore, C J; Johnson, Z P; Higgins, M; Toufexis, D; Wilson, M E

    2015-01-01

    Social subordination in macaque females is a known chronic stressor and previous studies have shown that socially subordinate female rhesus monkeys consume fewer kilocalories than dominant animals when a typical laboratory chow diet is available. However, in a rich dietary environment that provides access to chow in combination with a more palatable diet (i.e. high in fat and refined sugar), subordinate animals consume significantly more daily kilocalories than dominant conspecifics. Substantial literature is available supporting the role of stress hormone signals in shaping dietary preferences and promoting the consumption of palatable, energy-dense foods. The present study was conducted using stable groups of adult female rhesus monkeys to test the hypothesis that pharmacological treatment with a brain penetrable corticotrophin-releasing factor type 1 receptor (CRF1) antagonist would attenuate the stress-induced consumption of a palatable diet among subordinate animals in a rich dietary environment but would be without effect in dominant females. The results show that administration of the CRF1 receptor antagonist significantly reduced daily caloric intake of both available diets among subordinate females compared to dominant females. Importantly, multiple regression analyses showed that the attenuation in caloric intake in response to Antalarmin (Sigma-Aldrich, St Louis, MO, USA) was significantly predicted by the frequency of submissive and aggressive behaviour emitted by females, independent of social status. Taken together, the findings support the involvement of activation of CRF1 receptors in the stress-induced consumption of excess calories in a rich dietary environment and also support the growing literature concerning the importance of CRF for sustaining emotional feeding.

  5. Antagonism of Corticotrophin-Releasing Factor Type 1 Receptors Attenuates Caloric Intake of Free Feeding Subordinate Female Rhesus Monkeys in a Rich Dietary Environment

    PubMed Central

    Moore, C J; Johnson, Z P; Higgins, M; Toufexis, D; Wilson, M E

    2015-01-01

    Social subordination in macaque females is a known chronic stressor and previous studies have shown that socially subordinate female rhesus monkeys consume fewer kilocalories than dominant animals when a typical laboratory chow diet is available. However, in a rich dietary environment that provides access to chow in combination with a more palatable diet (i.e. high in fat and refined sugar), subordinate animals consume significantly more daily kilocalories than dominant conspecifics. Substantial literature is available supporting the role of stress hormone signals in shaping dietary preferences and promoting the consumption of palatable, energy-dense foods. The present study was conducted using stable groups of adult female rhesus monkeys to test the hypothesis that pharmacological treatment with a brain penetrable corticotrophin-releasing factor type 1 receptor (CRF1) antagonist would attenuate the stress-induced consumption of a palatable diet among subordinate animals in a rich dietary environment but would be without effect in dominant females. The results show that administration of the CRF1 receptor antagonist significantly reduced daily caloric intake of both available diets among subordinate females compared to dominant females. Importantly, multiple regression analyses showed that the attenuation in caloric intake in response to Antalarmin (Sigma-Aldrich, St Louis, MO, USA) was significantly predicted by the frequency of submissive and aggressive behaviour emitted by females, independent of social status. Taken together, the findings support the involvement of activation of CRF1 receptors in the stress-induced consumption of excess calories in a rich dietary environment and also support the growing literature concerning the importance of CRF for sustaining emotional feeding. PMID:25674637

  6. Genome characteristics of a novel phage from Bacillus thuringiensis showing high similarity with phage from Bacillus cereus.

    PubMed

    Yuan, Yihui; Gao, Meiying; Wu, Dandan; Liu, Pengming; Wu, Yan

    2012-01-01

    Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the "late" region, the "lysogeny-lysis" region and the "early" region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor.

  7. Genome Characteristics of a Novel Phage from Bacillus thuringiensis Showing High Similarity with Phage from Bacillus cereus

    PubMed Central

    Yuan, Yihui; Gao, Meiying; Wu, Dandan; Liu, Pengming; Wu, Yan

    2012-01-01

    Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the “late” region, the “lysogeny-lysis” region and the “early” region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor. PMID:22649540

  8. Mapping protease substrates using a biotinylated phage substrate library.

    SciTech Connect

    Scholle, M. D.; Kriplani, U.; Pabon, A.; Sishtla, K.; Glucksman, M. J.; Kay, B. K.; Biosciences Division; Chicago Medical School

    2005-05-05

    We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobic is the scissile bond.

  9. Palmitate-induced Endoplasmic Reticulum stress and subsequent C/EBPα Homologous Protein activation attenuates leptin and Insulin-like growth factor 1 expression in the brain.

    PubMed

    Marwarha, Gurdeep; Claycombe, Kate; Schommer, Jared; Collins, David; Ghribi, Othman

    2016-11-01

    The peptide hormones Insulin-like growth factor-1 (IGF1) and leptin mediate a myriad of biological effects - both in the peripheral and central nervous systems. The transcription of these two hormones is regulated by the transcription factor C/EBPα, which in turn is negatively regulated by the transcription factor C/EBP Homologous Protein (CHOP), a specific marker of endoplasmic reticulum (ER) stress. In the peripheral system, disturbances in leptin and IGF-1 levels are implicated in a variety of metabolic diseases including obesity, diabetes, atherosclerosis and cardiovascular diseases. Current research suggests a positive correlation between consumption of diets rich in saturated free fatty acids (sFFA) and metabolic diseases. Induction of ER stress and subsequent dysregulation in the expression levels of leptin and IGF-1 have been shown to mediate sFFA-induced metabolic diseases in the peripheral system. Palmitic acid (palmitate), the most commonly consumed sFFA, has been shown to be up-taken by the brain, where it may promote neurodegeneration. However, the extent to which palmitate induces ER stress in the brain and attenuates leptin and IGF1 expression has not been determined. We fed C57BL/6J mice a palmitate-enriched diet and determined effects on the expression levels of leptin and IGF1 in the hippocampus and cortex. We further determined the extent to which ER stress and subsequent CHOP activation mediate the palmitate effects on the transcription of leptin and IGF1. We demonstrate that palmitate induces ER stress and decreases leptin and IGF1 expression by inducing the expression of CHOP. The molecular chaperone 4-phenylbutyric acid (4-PBA), an inhibitor of ER stress, precludes the palmitate-evoked down-regulation of leptin and IGF1 expression. Furthermore, the activation of CHOP in response to ER stress is pivotal in the attenuation of leptin and IGF1 expression as knocking-down CHOP in mice or in SH-SY5Y and Neuro-2a (N2a) cells rescues the palmitate

  10. Attenuation of Nitrogen Mustard-Induced Pulmonary Injury and Fibrosis by Anti-Tumor Necrosis Factor-α Antibody.

    PubMed

    Malaviya, Rama; Sunil, Vasanthi R; Venosa, Alessandro; Verissimo, Vivianne L; Cervelli, Jessica A; Vayas, Kinal N; Hall, LeRoy; Laskin, Jeffrey D; Laskin, Debra L

    2015-11-01

    Nitrogen mustard (NM) is a bifunctional alkylating agent that causes acute injury to the lung that progresses to fibrosis. This is accompanied by a prominent infiltration of macrophages into the lung and upregulation of proinflammatory/profibrotic cytokines including tumor necrosis factor (TNF)α. In these studies, we analyzed the ability of anti-TNFα antibody to mitigate NM-induced lung injury, inflammation, and fibrosis. Treatment of rats with anti-TNFα antibody (15 mg/kg, iv, every 9 days) beginning 30 min after intratracheal administration of NM (0.125 mg/kg) reduced progressive histopathologic alterations in the lung including perivascular and peribronchial edema, macrophage/monocyte infiltration, interstitial thickening, bronchiolization of alveolar walls, fibrin deposition, emphysema, and fibrosis. NM-induced damage to the alveolar-epithelial barrier, measured by bronchoalveolar lavage (BAL) protein and cell content, was also reduced by anti-TNFα antibody, along with expression of the oxidative stress marker, heme oxygenase-1. Whereas the accumulation of proinflammatory/cytotoxic M1 macrophages in the lung in response to NM was suppressed by anti-TNFα antibody, anti-inflammatory/profibrotic M2 macrophages were increased or unchanged. Treatment of rats with anti-TNFα antibody also reduced NM-induced increases in expression of the profibrotic mediator, transforming growth factor-β. This was associated with a reduction in NM-induced collagen deposition in the lung. These data suggest that inhibiting TNFα may represent an efficacious approach to mitigating lung injury induced by mustards.

  11. Mutation within the hinge region of the transcription factor Nr2f2 attenuates salt-sensitive hypertension.

    PubMed

    Kumarasamy, Sivarajan; Waghulde, Harshal; Gopalakrishnan, Kathirvel; Mell, Blair; Morgan, Eric; Joe, Bina

    2015-01-01

    Genome-wide association studies (GWAS) have prioritized a transcription factor, nuclear receptor 2 family 2 (NR2F2), as being associated with essential hypertension in humans. Here we provide evidence that validates this association and indicates that Nr2f2 is a genetic determinant of blood pressure (BP). Using the zinc-finger nuclease technology, the generation of a targeted Nr2f2-edited rat model is reported. The resulting gene-edited rats have a 15 bp deletion in exon 2 leading to a five-amino-acid deletion in the hinge region of the mutant Nr2f2 protein. Both systolic and diastolic blood pressures of the Nr2f2(mutant) rats are significantly lower than controls. Because the hinge region of Nr2f2 is required for interaction with Friend of Gata2 (Fog2), protein-protein interaction is examined. Interaction of Nr2f2(mutant) protein with Fog2 is greater than that with the wild-type Nr2f2, indicating that the extent of interaction between these two transcription factors critically influences BP. PMID:25687237

  12. Sitagliptin attenuates inflammatory responses in lipopolysaccharide-stimulated cardiomyocytes via nuclear factor-κB pathway inhibition

    PubMed Central

    LIN, CHIEN-HUNG; LIN, CHUNG-CHING

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) and GLP-1 receptors (GLP-1Rs) are responsible for glucose homeostasis, and have been shown to reduce inflammation in preclinical studies. The aim of the present study was to determine whether sitagliptin, an inhibitor of the enzyme dipeptidyl peptidase-4 (DPP-4), as a GLP-1 receptor agonist, exerts an anti-inflammatory effect on cardiomyoblasts during lipopolysaccharide (LPS) stimulation. Exposure to LPS increased the expression levels of tumor necrosis factor (TNF)-α, interleukin-6 (IL)-6 and IL-1β in H9c2 cells, and also resulted in elevations in cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression and nuclear factor-κB (NF-κB) nuclear translocation. Treatment with the DPP-4 inhibitor sitagliptin dose-dependently downregulated the mRNA levels of IL-6, COX-2 and iNOS in LPS-stimulated H9c2 cells. In addition, sitagliptin inhibited the increased protein expression of IL-6, TNF-α and IL-1β. NF-κB mRNA expression was reduced and its translocation to the nucleus was suppressed by treatment with sitagliptin. The present results demonstrated that sitagliptin exerts a beneficial effect on cardiomyoblasts exposed to LPS by inhibiting expression of inflammatory mediators and suppressing NF-κB activation. These findings indicate that the DPP-4 inhibitor sitagliptin may serve a function in cardiac remodeling attributed to sepsis-induced inflammation. PMID:27284355

  13. Date syrup-derived polyphenols attenuate angiogenic responses and exhibits anti-inflammatory activity mediated by vascular endothelial growth factor and cyclooxygenase-2 expression in endothelial cells.

    PubMed

    Taleb, Hajer; Morris, R Keith; Withycombe, Cathryn E; Maddocks, Sarah E; Kanekanian, Ara D

    2016-07-01

    Bioactive components such as polyphenols, present in many plants, are purported to have anti-inflammatory and antiangiogenic properties. Date syrup, produced from date fruit of the date palm tree, has traditionally been used to treat a wide range of diseases with etiologies involving angiogenesis and inflammation. It was hypothesized that polyphenols in date syrup reduce angiogenic responses such as cell migration, tube formation, and matrix metalloproteinase activity in an inflammatory model by exhibiting anti-inflammatory activity mediated by vascular endothelial growth factor (VEGF) and the prostaglandin enzyme cyclooxygenase-2 (COX-2) in endothelial cells. Date syrup polyphenols at 60 and 600μg/mL reduced inflammation and suppressed several stages of angiogenesis, including endothelial cell migration, invasion, matrix metalloproteinase activity, and tube formation, without evidence of cytotoxicity. VEGF and COX-2 expression induced by tumor necrosis factor-alpha at both gene expression and protein level was significantly reduced by date syrup polyphenols in comparison to untreated cells. In conclusion, polyphenols in date syrup attenuated angiogenic responses and exhibited anti-inflammatory activity mediated by VEGF and COX-2 expression in endothelial cells. PMID:27333954

  14. Lactonase-expressing Lactobacillus plantarum NC8 attenuates the virulence factors of multiple drug resistant Pseudomonas aeruginosa in co-culturing environment.

    PubMed

    Joshi, Sudha; Kaur, Amanjot; Sharma, Prince; Harjai, Kusum; Capalash, Neena

    2014-08-01

    Pseudomonas aeruginosa possesses an arcade of both cell-associated and extracellular cytotoxic virulence factors which are regulated by a multi-component quorum sensing system. Many research studies report success of lactonase in combating the pathogenicity of P. aeruginosa but delivery of lactonase remains a challenge. The present study aims at developing a delivery vehicle for lactonase. Lactobacillus plantarum NC8 was used as host for aiiA (Bacillus thuringiensis 4A3 lactonase gene) using pSIP409 expression vector. pSIP409: aiiA construct was stably maintained in L. plantarum NC8. Co-culturing of multi-drug resistant (MDR) clinical isolates of P. aeruginosa and PAO1 with recombinant L. plantarum NC8 led to significant reduction (p < 0.001) in extracellular virulence factors like pyocyanin, protease, elastase and rhamnolipids in P. aeruginosa and also showed significant reduction in adhesion of P. aeruginosa strains to uroepithelial cells in vitro. This study shows the heterologous expression of AiiA lactonase in L. plantarum NC8. Co-culturing of lactonase expressing L. plantarum NC8 with MDR P. aeruginosa strains led to attenuation of their virulence significantly. These results underscore the potential application of recombinant L. plantarum NC8 with anti-quorum sensing properties to control infections caused by multidrug resistant P. aeruginosa.

  15. Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase.

    PubMed

    Jansen, P M; Pixley, R A; Brouwer, M; de Jong, I W; Chang, A C; Hack, C E; Taylor, F B; Colman, R W

    1996-03-15

    In previous studies, we have shown that administration of monoclonal antibody (MoAb) C6B7 against human factor XII to baboons challenged with a lethal dose of Escherichia coli abrogates activation of the contact system and modulates secondary hypotension. To evaluate the contribution of activated contact proteases to the appearance of other inflammatory mediators in this experimental model of sepsis, we studied the effect of administration of MoAb C6B7 on activation of complement and fibrinolytic cascades, stimulation of neutrophil degranulation, and release of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Activation of the complement system, as reflected by circulating C3b/c and C4b/c levels, was significantly reduced in five animals that had received MoAb C6B7 before a lethal dose of E coli as compared with five control animals that had been given a lethal challenge only. Inhibition of contact activation also modulated the fibrinolytic response, since the release of tissue-type plasminogen activator (t-PA) and the appearance of plasmin-alpha2-antiplasmin (PAP) complexes into the circulation was significantly attenuated upon pretreatment with anti-factor XII MoAb. In contrast, plasma levels of plasminogen activator inhibitor (PAI) were modestly enhanced in the treatment group. Degranulation of neutrophils, as assessed by circulating elastase-alpha1-protease inhibitor complexes, and release of IL-6 but not of TNF-alpha was decreased in anti-factor XII-treated animals. Observed differences in the inflammatory response between treatment and control groups were not likely due to different challenges, since the number of E coli that had been infused, as well as circulating levels of endotoxin after the challenge, were similar for both groups. These data suggest that activation of the contact system modulates directly or indirectly various mediator systems involved in the inflammatory response during severe sepsis in

  16. WE-E-18A-05: Bremsstrahlung of Laser-Plasma Interaction at KeV Temperature: Forward Dose and Attenuation Factors

    SciTech Connect

    Saez-Beltran, M; Fernandez Gonzalez, F

    2014-06-15

    Purpose: To obtain an analytical empirical formula for the photon dose source term in forward direction from bremsstrahlung generated from laser-plasma accelerated electron beams in aluminum solid targets, with electron-plasma temperatures in the 10–100 keV energy range, and to calculate transmission factors for iron, aluminum, methacrylate, lead and concrete and air, materials most commonly found in vacuum chamber labs. Methods: Bremsstrahlung fluence is calculated from the convolution of thin-target bremsstrahlung spectrum for monoenergetic electrons and the relativistic Maxwell-Juettner energy distribution for the electron-plasma. Unattenuatted dose in tissue is calculated by integrating the photon spectrum with the mass-energy absorption coefficient. For the attenuated dose, energy dependent absorption coefficient, build-up factors and finite shielding correction factors were also taken into account. For the source term we use a modified formula from Hayashi et al., and we fitted the proportionality constant from experiments with the aid of the previously calculated transmission factors. Results: The forward dose has a quadratic dependence on electron-plasma temperature: 1 joule of effective laser energy transferred to the electrons at 1 m in vacuum yields 0,72 Sv per MeV squared of electron-plasma temperature. Air strongly filters the softer part of the photon spectrum and reduce the dose to one tenth in the first centimeter. Exponential higher energy tail of maxwellian spectrum contributes mainly to the transmitted dose. Conclusion: A simple formula for forward photon dose from keV range temperature plasma is obtained, similar to those found in kilovoltage x-rays but with higher dose per dissipated electron energy, due to thin target and absence of filtration.

  17. Phage on tap–a quick and efficient protocol for the preparation of bacteriophage laboratory stocks

    PubMed Central

    Bonilla, Natasha; Rojas, Maria Isabel; Netto Flores Cruz, Giuliano; Hung, Shr-Hau; Rohwer, Forest

    2016-01-01

    A major limitation with traditional phage preparations is the variability in titer, salts, and bacterial contaminants between successive propagations. Here we introduce the Phage On Tap (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) stocks. This method produces homogenous, laboratory-scale, high titer (up to 1010–11 PFU·ml−1), endotoxin reduced phage banks that can be used to eliminate the variability between phage propagations and improve the molecular characterizations of phage. The method consists of five major parts, including phage propagation, phage clean up by 0.22 μm filtering and chloroform treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and storage of phage banks for continuous laboratory use. From a starting liquid lysate of > 100 mL, the PoT protocol generated a clean, homogenous, laboratory phage bank with a phage recovery efficiency of 85% within just two days. In contrast, the traditional method took upwards of five days to produce a high titer, but lower volume phage stock with a recovery efficiency of only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing endotoxin concentrations by over 3,000-fold while maintaining phage titer. The PoT protocol focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated to sufficient titer, producing homogenous, high titer phage banks that are applicable for molecular and cellular assays. PMID:27547567

  18. Phage on tap-a quick and efficient protocol for the preparation of bacteriophage laboratory stocks.

    PubMed

    Bonilla, Natasha; Rojas, Maria Isabel; Netto Flores Cruz, Giuliano; Hung, Shr-Hau; Rohwer, Forest; Barr, Jeremy J

    2016-01-01

    A major limitation with traditional phage preparations is the variability in titer, salts, and bacterial contaminants between successive propagations. Here we introduce the Phage On Tap (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) stocks. This method produces homogenous, laboratory-scale, high titer (up to 10(10-11) PFU·ml(-1)), endotoxin reduced phage banks that can be used to eliminate the variability between phage propagations and improve the molecular characterizations of phage. The method consists of five major parts, including phage propagation, phage clean up by 0.22 μm filtering and chloroform treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and storage of phage banks for continuous laboratory use. From a starting liquid lysate of > 100 mL, the PoT protocol generated a clean, homogenous, laboratory phage bank with a phage recovery efficiency of 85% within just two days. In contrast, the traditional method took upwards of five days to produce a high titer, but lower volume phage stock with a recovery efficiency of only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing endotoxin concentrations by over 3,000-fold while maintaining phage titer. The PoT protocol focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated to sufficient titer, producing homogenous, high titer phage banks that are applicable for molecular and cellular assays. PMID:27547567

  19. Development of phage/antibody immobilized magnetostrictive biosensors

    NASA Astrophysics Data System (ADS)

    Fu, Liling

    There is an urgent need for biosensors that are able to detect and quantify the presence of a small amount of pathogens in a real-time manner accurately and quickly to guide prevention efforts and assay food and water quality. Acoustic wave (AW) devices, whose performance is defined by mass sensitivity (Sm) and quality factor (Q value), have been extensively studied as high performance biosensor platforms. However, current AW devices still face some challenges such as the difficulty to be employed in liquid and low Q value in practical applications. The objective of this research is to develop magnetostrictive sensors which include milli/microcantilever type (MSMC) and particle type (MSP). Compared to other AW devices, MSMC exhibits the following advantages: (1) wireless/remote driving and sensing; (2) easy to fabricate; (3) works well in liquid; (4) exhibits a high Q value (> 500 in air). The fundamental study of the damping effect on MSMCs from the surrounding media including air and liquids were conducted to improve the Q value of MSMCs. The experiment results show that the Q value is dependent on the properties of surrounding media (e.g. viscosity, density), the geometry of the MSMCs, and the harmonic mode on the resonance behavior of MSMCs, etc. The phage-coated MSMC has high specificity and sensitivity even while used in water with a low concentration of targeted bacteria. Two currently developed phages, JRB7 and E2, respectively respond to Bacillus anthracis spores and Salmonella typhimurium, were employed as bio-recognition elements in this research. The phage-immobilized MSMC biosensors exhibited high performance and detection of limit was 5 x 104 cfu/ml for the MSMC in size of 1.4 x 0.8 x 0.035 mm. The MSMC-based biosensors were indicated as a very potential method for in-situ monitoring of the biological quality in water. The MSP combine antibody was used to detect Staphylococcus aureus in this experiment. The interface between MSPs and antibody was

  20. Cryptic transposable phages of Pseudomonas aeruginosa

    SciTech Connect

    Krylov, V.N.; Mit`kina, L.N.; Pleteneva, E.A.; Aleshin, V.V.

    1995-11-01

    Frequencies of nucleotide sequences homologous to phage transposons (PT) of two species, D3112 and B3, were assessed in genomes of natural Pseudomonas aeruginosa strains by the dot-blot hybridization method. These strains were incapable of liberating viable phages on a lawn of the PA01 standard indicator strain of P. aeruginosa. It was shown that the homologies detected belong to two groups, high and intermediate, with respect to homology level. Homology patterns were classified as high when they provided signals comparable to those for hybridization in a positive control; patterns were classified as intermediate when the hybridization level was higher than the background level, but lower than in the positive control. Homologous PT sequences were designated as cryptic PT. Intact cryptic PT prophages were shown to exist in genomes of particular natural strains manifesting a higher level of hybridization. However, the growth of these phages was limited by the restriction system of strain PA01. It is possible to isolate strains maintaining the growth of some cryptic PT. These strains differed from P. aeruginosa with respect to the specificity of the restriction and modification system. Nevertheless, in most cases, the attempt to identify a novel host capable of maintaining growth of a cryptic PT failed. Natural strains often carry cryptic PT related to both known PT species, D3112 and B3. The frequency of cryptic PT is extremely high, reaching 30% in strains with a high level of homology only and up to 50% in all strains exhibiting homology. This high PT frequency is assumed to be associated with the considerable variation of P. aeruginosa. 15 refs., 1 fig., 2 tabs.

  1. MIIP accelerates epidermal growth factor receptor protein turnover and attenuates proliferation in non-small cell lung cancer

    PubMed Central

    Wen, Jing; Fu, Jianhua; Ling, Yihong; Zhang, Wei

    2016-01-01

    The migration and invasion inhibitory protein (MIIP) has been discovered recently to have inhibitory functions in cell proliferation and migration. Overexpression of MIIP reduced the intracellular steady-state level of epidermal growth factor receptor (EGFR) protein in lung cancer cells with no effect on EGFR mRNA expression compared to that in the control cells. This MIIP-promoted EGFR protein degradation was reversed by proteasome and lysosome inhibitors, suggesting the involvement of both proteasomal and lysosomal pathways in this degradation. This finding was further validated by pulse-chase experiments using 35S-methionine metabolic labeling. We found that MIIP accelerates EGFR protein turnover via proteasomal degradation in the endoplasmic reticulum and then via the lysosomal pathway after its entry into endocytic trafficking. MIIP-stimulated downregulation of EGFR inhibits downstream activation of Ras and blocks the MEK signal transduction pathway, resulting in inhibition of cell proliferation. The negative correlation between MIIP and EGFR protein expression was validated in lung adenocarcinoma samples. Furthermore, the higher MIIP protein expression predicts a better overall survival of Stage IA-IIIA lung adenocarcinoma patients who underwent radical surgery. These findings reveal a new mechanism by which MIIP inhibits cell proliferation. PMID:26824318

  2. Doxazosin Treatment Attenuates Carbon Tetrachloride-Induced Liver Fibrosis in Hamsters through a Decrease in Transforming Growth Factor β Secretion

    PubMed Central

    Muñoz-Ortega, Martin Humberto; Llamas-Ramírez, Raúl Wiliberto; Romero-Delgadillo, Norma Isabel; Elías-Flores, Tania Guadalupe; de Jesus Tavares-Rodríguez, Edgar; del Rosario Campos-Esparza, María; Cervantes-García, Daniel; Muñoz-Fernández, Luis; Gerardo-Rodríguez, Martin; Ventura-Juárez, Javier

    2016-01-01

    Background/Aims The development of therapeutic strategies for the treatment of cirrhosis has become an important focus for basic and clinical researchers. Adrenergic receptor antagonists have been evaluated as antifibrotic drugs in rodent models of carbon tetrachloride (CCl4)-induced cirrhosis. The aim of the present study was to evaluate the effects of carvedilol and doxazosin on fibrosis/cirrhosis in a hamster animal model. Methods Cirrhotic-induced hamsters were treated by daily administration of carvedilol and doxazosin for 6 weeks. Hepatic function and histological evaluation were conducted by measuring biochemical markers, including total bilirubin, aspartate aminotransferase, alanine aminotransferase and albumin, and liver tissue slices. Additionally, transforming growth factor β (TGF-β) immunohistochemistry was analyzed. Results Biochemical markers revealed that hepatic function was restored after treatment with doxazosin and carvedilol. Histological evaluation showed a decrease in collagen type I deposits and TGF-β-secreting cells. Conclusions Taken together, these results suggest that the decrease in collagen type I following treatment with doxazosin or carvedilol is achieved by decreasing the profibrotic activities of TGF-β via the blockage of α1- and β-adrenergic receptor. Consequently, a diminution of fibrotic tissue in the CCl4-induced model of cirrhosis is achieved. PMID:26573293

  3. Loss of CEACAM1, a Tumor-Associated Factor, Attenuates Post-infarction Cardiac Remodeling by Inhibiting Apoptosis

    PubMed Central

    Wang, Yan; Chen, Yanmei; Yan, Yi; Li, Xinzhong; Chen, Guojun; He, Nvqin; Shen, Shuxin; Chen, Gangbin; Zhang, Chuanxi; Liao, Wangjun; Liao, Yulin; Bin, Jianping

    2016-01-01

    Carcinoembryonic antigen-related cell adhesion molecule1 (CEACAM1) is a tumor-associated factor that is known to be involved in apoptosis, but the role of CEACAM1 in cardiovascular disease is unclear. We aims to investigate whether CEACAM1 influences cardiac remodeling in mice with myocardial infarction (MI) and hypoxia-induced cardiomyocyte injury. Both serum in patients and myocardial CEACAM1 levels in mice were significantly increased in response to MI, while levels were elevated in neonatal rat cardiomyocytes (NRCs) exposed to hypoxia. Eight weeks after MI, a lower mortality rate, improved cardiac function, and less cardiac remodeling in CEACAM1 knock-out (KO) mice than in their wild-type (WT) littermates were observed. Moreover, myocardial expression of mitochondrial Bax, cytosolic cytochrome C, and cleaved caspase-3 was significantly lower in CEACAM1 KO mice than in WT mice. In cultured NRCs exposed to hypoxia, recombinant human CEACAM1 (rhCEACAM1) reduced mitochondrial membrane potential, upregulated mitochondrial Bax, increased cytosolic cytochrome C and cleaved caspase-3, and consequently increased apoptosis. RhCEACAM1 also increased the levels of GRP78 and CHOP in NRCs with hypoxia. All of these effects were abolished by silencing CEACAM1. Our study indicates that CEACAM1 exacerbates hypoxic cardiomyocyte injury and post-infarction cardiac remodeling by enhancing cardiomyocyte mitochondrial dysfunction and endoplasmic reticulum stress-induced apoptosis. PMID:26911181

  4. Mycobacterium leprae-induced Insulin-like Growth Factor I attenuates antimicrobial mechanisms, promoting bacterial survival in macrophages

    PubMed Central

    Batista-Silva, L. R.; Rodrigues, Luciana Silva; Vivarini, Aislan de Carvalho; Costa, Fabrício da Mota Ramalho; Mattos, Katherine Antunes de; Costa, Maria Renata Sales Nogueira; Rosa, Patricia Sammarco; Toledo-Pinto, T. G.; Dias, André Alves; Moura, Danielle Fonseca; Sarno, Euzenir Nunes; Lopes, Ulisses Gazos; Pessolani, Maria Cristina Vidal

    2016-01-01

    Mycobacterium leprae (ML), the etiologic agent of leprosy, can subvert macrophage antimicrobial activity by mechanisms that remain only partially understood. In the present study, the participation of hormone insulin-like growth factor I (IGF-I) in this phenomenum was investigated. Macrophages from the dermal lesions of the disseminated multibacillary lepromatous form (LL) of leprosy expressed higher levels of IGF-I than those from the self-limited paucibacillary tuberculoid form (BT). Higher levels of IGF-I secretion by ML-infected macrophages were confirmed in ex vivo and in vitro studies. Of note, the dampening of IGF-I signaling reverted the capacity of ML-infected human and murine macrophages to produce antimicrobial molecules and promoted bacterial killing. Moreover, IGF-I was shown to inhibit the JAK/STAT1-dependent signaling pathways triggered by both mycobacteria and IFN-γ most probably through its capacity to induce the suppressor of cytokine signaling-3 (SOCS3). Finally, these in vitro findings were corroborated by in vivo observations in which higher SOCS3 expression and lower phosphorylation of STAT1 levels were found in LL versus BT dermal lesions. Altogether, our data strongly suggest that IGF-I contributes to the maintenance of a functional program in infected macrophages that suits ML persistence in the host, reinforcing a key role for IGF-I in leprosy pathogenesis. PMID:27282338

  5. Platelet-derived growth factor-D modulates extracellular matrix homeostasis and remodeling through TIMP-1 induction and attenuation of MMP-2 and MMP-9 gelatinase activities

    SciTech Connect

    Borkham-Kamphorst, Erawan Alexi, Pascal; Tihaa, Lidia; Haas, Ute; Weiskirchen, Ralf

    2015-02-13

    Platelet-derived growth factor-D (PDGF-D) is a more recent recognized growth factor involved in the regulation of several cellular processes, including cell proliferation, transformation, invasion, and angiogenesis by binding to and activating its cognate receptor PDGFR-β. After bile duct ligation or in the carbon tetrachloride-induced hepatic fibrosis model{sub ,} PDGF-D showed upregulation comparable to PDGF-B. Moreover, adenoviral PDGF-D gene transfer induced hepatic stellate cell proliferation and liver fibrosis. We here investigated the molecular mechanism of PDGF-D involvement in liver fibrogenesis. Therefore, the GRX mouse cell line was stimulated with PDGF-D and evaluated for fibrotic markers and PDGF-D signaling pathways in comparison to the other PDGF isoforms. We found that PDGF-D failed to enhance Col I and α-smooth muscle actin (α-SMA) production but has capacity to upregulate expression of the tissue inhibitor of metalloprotease 1 (TIMP-1) resulting in attenuation of MMP-2 and MMP-9 gelatinase activity as indicated by gelatinase zymography. This phenomenon was restored through application of a PDGF-D neutralizing antibody. Unexpectedly, PDGF-D incubation decreased both PDGFR-α and -β in mRNA and protein levels, and PDGF-D phosphorylated typrosines specific for PDGFR-α and -β. We conclude that PDGF-D intensifies fibrogenesis by interfering with the fibrolytic activity of the TIMP-1/MMP system and that PDGF-D signaling is mediated through both PDGF-α and -β receptors. - Highlights: • PDGF-D signals through PDGF receptor type α and β. • PDGF-D modulates extracellular matrix homeostasis and remodeling. • Like PDGF-B, PDGF-D triggers phosphorylation of PLC-γ, Akt/PKB, JNK, ERK1/2, and p38. • PDGF-D induces TIMP-1 expression through ERK and p38 MAPK. • PDGF-D attenuates MMP-2 and MMP-9 gelatinase activities.

  6. DJ-1 upregulates anti-oxidant enzymes and attenuates hypoxia/re-oxygenation-induced oxidative stress by activation of the nuclear factor erythroid 2-like 2 signaling pathway.

    PubMed

    Yan, Yu-Feng; Yang, Wen-Jie; Xu, Qiang; Chen, He-Ping; Huang, Xiao-Shan; Qiu, Ling-Yu; Liao, Zhang-Ping; Huang, Qi-Ren

    2015-09-01

    DJ-1 protein, as a multifunctional intracellular protein, has an important role in transcriptional regulation and anti-oxidant stress. A recent study by our group showed that DJ-1 can regulate the expression of certain anti‑oxidant enzymes and attenuate hypoxia/re‑oxygenation (H/R)‑induced oxidative stress in the cardiomyocyte cell line H9c2; however, the detailed molecular mechanisms have remained to be elucidated. Nuclear factor erythroid 2‑like 2 (Nrf2) is an essential transcription factor that regulates the expression of several anti‑oxidant genes via binding to the anti‑oxidant response element (ARE). The present study investigated whether activation of the Nrf2 pathway is responsible for the induction of anti‑oxidative enzymes by DJ‑1 and contributes to the protective functions of DJ‑1 against H/R‑induced oxidative stress in H9c2 cells. The results demonstrated that DJ‑1‑overexpressing H9c2 cells exhibited anti‑oxidant enzymes, including manganese superoxide dismutase, catalase and glutathione peroxidase, to a greater extent and were more resistant to H/R‑induced oxidative stress compared with native cells, whereas DJ‑1 knockdown suppressed the induction of these enzymes and further augmented the oxidative stress injury. Determination of the importance of Nrf2 in DJ‑1‑mediated anti‑oxidant enzymes induction and cytoprotection against oxidative stress induced by H/R showed that overexpression of DJ‑1 promoted the dissociation of Nrf2 from its cytoplasmic inhibitor Keap1, resulting in enhanced levels of nuclear translocation, ARE‑binding and transcriptional activity of Nrf2. Of note, Nrf2 knockdown abolished the DJ‑1‑mediated induction of anti‑oxidant enzymes and cytoprotection against oxidative stress induced by H/R. In conclusion, these findings indicated that activation of the Nrf2 pathway is a critical mechanism by which DJ-1 upregulates anti-oxidative enzymes and attenuates H/R-induced oxidative stress in H9c2

  7. Strain Specific Phage Treatment for Staphylococcus aureus Infection Is Influenced by Host Immunity and Site of Infection

    PubMed Central

    Pincus, Nathan B.; Jammeh, Momodou L.; Datta, Sandip K.; Myles, Ian A.

    2015-01-01

    The response to multi-drug resistant bacterial infections must be a global priority. While mounting resistance threatens to create what the World Health Organization has termed a “post-antibiotic era”, the recent discovery that antibiotic use may adversely impact the microbiome adds further urgency to the need for new developmental approaches for anti-pathogen treatments. Methicillin-resistant Staphylococcus aureus (MRSA), in particular, has declared itself a serious threat within the United States and abroad. A potential solution to the problem of antibiotic resistance may not entail looking to the future for completely novel treatments, but instead looking into our history of bacteriophage therapy. This study aimed to test the efficacy, safety, and commercial viability of the use of phages to treat Staphylococcus aureus infections using the commercially available phage SATA-8505. We found that SATA-8505 effectively controls S. aureus growth and reduces bacterial viability both in vitro and in a skin infection mouse model. However, this killing effect was not observed when phage was cultured in the presence of human whole blood. SATA-8505 did not induce inflammatory responses in peripheral blood mononuclear cultures. However, phage did induce IFN gamma production in primary human keratinocyte cultures and induced inflammatory responses in our mouse models, particularly in a mouse model of chronic granulomatous disease. Our findings support the potential efficacy of phage therapy, although regulatory and market factors may limit its wider investigation and use. PMID:25909449

  8. A novel transducible chimeric phage from Escherichia coli O157:H7 Sakai strain encoding Stx1 production.

    PubMed

    Sváb, Domonkos; Bálint, Balázs; Maróti, Gergely; Tóth, István

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC), and especially enterohaemorrhagic E. coli (EHEC) are important, highly virulent zoonotic and food-borne pathogens. The genes encoding their key virulence factors, the Shiga toxins, are distributed by converting bacteriophages, the Stx phages. In this study we isolated a new type of inducible Stx phage carrying the stx1 gene cluster from the prototypic EHEC O157:H7 Sakai strain. The phage showed Podoviridae morphology, and was capable of converting the E. coli K-12 MG1655 strain to Shiga toxin-producing phenotype. The majority of the phage genes originate from the stx2-encoding Sakai prophage Sp5, with major rearrangements in its genome. Beside certain minor recombinations, the genomic region originally containing the stx2 genes in Sp5 was replaced by a region containing six open reading frames from prophage Sp15 including stx1 genes. The rearranged genome, together with the carriage of stx1 genes, the morphology and the capability of lysogenic conversion represent a new type of recombinant Stx1 converting phage from the Sakai strain. PMID:25445656

  9. Pseudomonas aeruginosa adaptation to lungs of cystic fibrosis patients leads to lowered resistance to phage and protist enemies.

    PubMed

    Friman, Ville-Petri; Ghoul, Melanie; Molin, Søren; Johansen, Helle Krogh; Buckling, Angus

    2013-01-01

    Pathogenic life styles can lead to highly specialized interactions with host species, potentially resulting in fitness trade-offs in other ecological contexts. Here we studied how adaptation of the environmentally transmitted bacterial pathogen, Pseudomonas aeruginosa, to cystic fibrosis (CF) patients affects its survival in the presence of natural phage (14/1, ΦKZ, PNM and PT7) and protist (Tetrahymena thermophila and Acanthamoebae polyphaga) enemies. We found that most of the bacteria isolated from relatively recently intermittently colonised patients (1-25 months), were innately phage-resistant and highly toxic for protists. In contrast, bacteria isolated from long time chronically infected patients (2-23 years), were less efficient in both resisting phages and killing protists. Moreover, chronic isolates showed reduced killing of wax moth larvae (Galleria mellonella) probably due to weaker in vitro growth and protease expression. These results suggest that P. aeruginosa long-term adaptation to CF-lungs could trade off with its survival in aquatic environmental reservoirs in the presence of microbial enemies, while lowered virulence could reduce pathogen opportunities to infect insect vectors; factors that are both likely to result in poorer environmental transmission. From an applied perspective, phage therapy could be useful against chronic P. aeruginosa lung infections that are often characterized by multidrug resistance: chronic isolates were least resistant to phages and their poor growth will likely slow down the emergence of beneficial resistance mutations.

  10. Electron microscopy of virulent phages for Streptococcus lactis.

    PubMed Central

    Tsaneva, K P

    1976-01-01

    Electron microscopic studies were made on eight virulent Streptococcus lactis bacteriophages. These phages were taken as representative of eight host range groups established in a study of 75 phage isolates and 253 hosts (213 S. lactis, 22 S. cremoris, 18 S. diacetilactis). The phages studied were shown to have an isometric hexagonal head and noncontractile tails, usually several times longer than the head diameter. The virus heads were octahedral. The phages investigated represented three morphological types on the basis of head diameter , tail thickness, and tail length. These dimensions were approximately: for type I phages, 63, 172, and 11 nm, respectively; type II, 73, 200, and 20 nm, respectively; and type III, represented here by a single phage, 98, 551, and 12 nm, respectively. The tail surface revealed a different arrangment of the structural subunits which lent a helical appearance to the tails of type I and II phages and a guaffered tube appearance to the tail of type III phage. The number of turns along the tail axis, turn length, axial pitch, and helix angle were: type I, 32, 12 to 13 nm, 7.14 nm, and 11 degrees 43', respectively; type II, 24, 24, to 28 nm, 40.00 nm, and 32 degrees 30', respectively; and type III, 120, 12 nm, and no visible slope towards the axis. The morphology types showed complete correlation with serological groups, but not with groups based on host range pattern. Images PMID:817668

  11. Phage DNA dynamics in cells with different fates.

    PubMed

    Shao, Qiuyan; Hawkins, Alexander; Zeng, Lanying

    2015-04-21

    Bacteriophage λ begins its infection cycle by ejecting its DNA into its host Escherichia coli cell, after which either a lytic or a lysogenic pathway is followed, resulting in different cell fates. In this study, using a new technique to monitor the spatiotemporal dynamics of the phage DNA in vivo, we found that the phage DNA moves via two distinct modes, localized motion and motion spanning the whole cell. One or the other motion is preferred, depending on where the phage DNA is ejected into the cell. By examining the phage DNA trajectories, we found the motion to be subdiffusive. Moreover, phage DNA motion is the same in the early phase of the infection cycle, irrespective of whether the lytic or lysogenic pathway is followed; hence, cell-fate decision-making appears not to be correlated with the phage DNA motion. However, after the cell commits to one pathway or the other, phage DNA movement slows during the late phase of the lytic cycle, whereas it remains the same during the entire lysogenic cycle. Throughout the infection cycle, phage DNA prefers the regions around the quarter positions of the cell. PMID:25902444

  12. Computational models of populations of bacteria and lytic phage.

    PubMed

    Krysiak-Baltyn, Konrad; Martin, Gregory J O; Stickland, Anthony D; Scales, Peter J; Gras, Sally L

    2016-11-01

    The use of phages to control and reduce numbers of unwanted bacteria can be traced back to the early 1900s, when phages were explored as a tool to treat infections before the wide scale use of antibiotics. Recently, phage therapy has received renewed interest as a method to treat multiresistant bacteria. Phages are also widely used in the food industry to prevent the growth of certain bacteria in foods, and are currently being explored as a tool for use in bioremediation and wastewater treatment. Despite the large body of biological research on phages, relatively little attention has been given to computational modeling of the population dynamics of phage and bacterial interactions. The earliest model was described by Campbell in the 1960s. Subsequent modifications to this model include partial or complete resistance, multiple phage binding sites, and spatial heterogeneity. This review provides a general introduction to modeling of the population dynamics of bacteria and phage. The review introduces the basic model and relevant concepts and evaluates more complex variations of the basic model published to date, including a model of disease epidemics caused by infectious bacteria. Finally, the shortcomings and potential ways to improve the models are discussed.

  13. Detection of TNT-derivatives with recombinant phages

    PubMed Central

    Simonovicá, Mladen; Simonovicá, Branislav

    2012-01-01

    New immunoreagents for detection of TNT-derivatives TNP and TNP-Tris were developed using phage display technique. The monovalent and pentavalent recombinant phages carrying scFv specific for TNT were constructed and compared with each other to define the impact of valency and molecule dimension on antibody binding in immunoassay. Also, the bifunctional phages were generated, which carried TNT-specific scFvs as well as enzyme β-lactamase as a model marker on its surface. The most sensitive recombinant phages were selected and used for detection of TNP-Tris in a competitive ELISA based on immobilized antigen. Preincubation and partial phages saturation with a sample containing antigen allowed competition with immobilized hapten and displacement of free antigen. The phages exposing enzyme were used as immunoreagents for single step detection. The other phages were detected with specific marked antibodies. To date, the results presented in this paper are the first ever published regarding the recombinant phages for the detection of TNT. PMID:23050216

  14. High frequency generalized transduction by miniMu plasmid phage.

    PubMed

    Wang, B M; Liu, L; Groisman, E A; Casadaban, M J; Berg, C M

    1987-06-01

    Deletion derivatives of phage Mu which replicate as multicopy plasmids, and also transpose and package like Mu, have been developed for the in vivo cloning of bacterial genes. We show here that these miniMu plasmid phage are also efficient at generalized transduction and that both in vivo cloning and generalized transduction of a given gene can be accomplished in a single experiment.

  15. Calpain Inhibitor PD150606 Attenuates Glutamate Induced Spiral Ganglion Neuron Apoptosis through Apoptosis Inducing Factor Pathway In Vitro

    PubMed Central

    Song, Yong-Li; Chen, Xiao-Dong; Mi, Wen-Juan; Wang, Jian; Lin, Ying; Chen, Fu-Quan; Qiu, Jian-Hua

    2015-01-01

    Objective This research aimed to investigate whether glutamate induced spiral ganglion neurons (SGNs) apoptosis through apoptosis inducing factor (AIF) pathway. And verify whether PD150606, a calpain inhibitor could prevent apoptosis by inhibiting cleaving and releasing AIF in mitochondrion. Methods SGNs of postnatal days 0-3 were harvested and cultured in dishes. 20 mM Glu, the caspase inhibitor Z-VAD-FMK and calpain inhibitor PD150606 were added into cultured dishes separately. We used optical microscope and immunofluoresence staining to observe cell morphology and AIF distribution, RT-PCR and Westernblot to analyse AIF and calpain expression in SGNs. TUNEL assay was used to test cell apoptosis. Results Cell morphology and nuclear translocation of AIF were altered in SGNs by 20 mM Glu treated in vitro. The axon of SGN shortened, more apoptosis SGN were observed and the expression of AIF and calpain were up-regulated in Glu-treated group than the normal one (P<0.05). The same experiments were conducted in 20 mM+PD150606 treated group and 20 mM+Z-VAD-FMK group. Obviously AIF were located from cytoplasm to the nuclear and the expressions of AIF and calpain were down-regulated by PD150606 (P<0.05). Positive cells in TUNEL staining decreased after PD150606 treating. However, Z-VAD-FMK had no influence on AIF, calpain expression or cell apoptosis. Conclusion The AIF-related apoptosis pathway is involved in the process of Glu-induced SGN injury. Furthermore, the inhibition of calpain can prevent AIF from releasing the nuclear or inducing SGN apoptosis. PMID:25874633

  16. Blockade of Ets-1 attenuates epidermal growth factor-dependent collagen loss in human carotid plaque smooth muscle cells.

    PubMed

    Rao, Velidi H; Rai, Vikrant; Stoupa, Samantha; Agrawal, Devendra K

    2015-09-15

    Although degradation of extracellular matrix by matrix metalloproteinases (MMPs) is thought to be involved in symptomatic (S) carotid plaques in atherosclerosis, the mechanisms of MMP expression are poorly understood. Here, we demonstrate that collagen loss in vascular smooth vessel cells (VSMCs) isolated from S plaques was induced by epidermal growth factor (EGF) through the activation of p38-MAPK and JNK-MAPK pathways. Inhibitors of p38-MAPK and JNK-MAPK signaling pathways downregulated the expression of MMP-1 and MMP-9. In addition, we examined whether v-ets erythroblastosis virus E26 oncogene homologue 1 (Ets-1), an important regulator of different genes, is involved in destabilizing S plaques in patients with carotid stenosis. We demonstrate that EGF induces Ets-1 expression and decreases interstitial and basement membrane collagen in vascular smooth muscle cells (VSMCs) from patients with carotid stenosis. Increased expression of MMP-1 and -9 and decreased collagen mRNA transcripts were also found in Ets-1-overexpressed VSMCs. Transfection with both dominant-negative form of Ets-1 and small interfering RNA blocked EGF-induced MMP-1 and -9 expressions and increased the mRNA transcripts for collagen I (α1) and collagen III (α1) in S compared with asymptomatic (AS) carotid plaques. Inhibitors of p38-MAPK (SB202190) and JNK-MAPK (SP600125) signaling pathways decreased the expression of Ets-1, MMP-1, and MMP-9 and increased collagen type I and III expression in EGF-treated VSMCs. This study provides a mechanistic insight into the role of Ets-1 in the plaque destabilization in patients with carotid stenosis involving p38-MAPK and JNK signaling pathways.

  17. von Willebrand factor (VWF) propeptide binding to VWF D′D3 domain attenuates platelet activation and adhesion

    PubMed Central

    Madabhushi, Sri R.; Shang, Chengwei; Dayananda, Kannayakanahalli M.; Rittenhouse-Olson, Kate; Murphy, Mary; Ryan, Thomas E.; Montgomery, Robert R.

    2012-01-01

    Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D′D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D′D3 domain. At pH 6.2 and 10mM Ca2+, conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (KD = 0.2nM, koff = 8 × 10−5 s−1). Significant, albeit weaker, binding (KD = 25nM, koff = 4 × 10−3 s−1) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca2+. This interaction was also observed in human plasma (KD = 50nM). The addition of recombinant VWFpp in both flow-chamber–based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D′D3 mAb DD3.1, which blocks VWFpp binding to VWF-D′D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα. PMID:22452980

  18. Phages preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: past, present and future.

    PubMed

    Gillis, Annika; Mahillon, Jacques

    2014-07-01

    Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here.

  19. Phages preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: past, present and future.

    PubMed

    Gillis, Annika; Mahillon, Jacques

    2014-07-01

    Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here. PMID:25010767

  20. Phages Preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: Past, Present and Future

    PubMed Central

    Gillis, Annika; Mahillon, Jacques

    2014-01-01

    Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here. PMID:25010767

  1. Fibroblast Growth Factor-9 Enhances M2 Macrophage Differentiation and Attenuates Adverse Cardiac Remodeling in the Infarcted Diabetic Heart

    PubMed Central

    Singla, Dinender K.; Singla, Reetu D.; Abdelli, Latifa S.; Glass, Carley

    2015-01-01

    Inflammation has been implicated as a perpetrator of diabetes and its associated complications. Monocytes, key mediators of inflammation, differentiate into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages upon infiltration of damaged tissue. However, the inflammatory cell types, which propagate diabetes progression and consequential adverse disorders, remain unclear. The current study was undertaken to assess monocyte infiltration and the role of fibroblast growth factor-9 (FGF-9) on monocyte to macrophage differentiation and cardioprotection in the diabetic infarcted heart. Db/db diabetic mice were assigned to sham, myocardial infarction (MI), and MI+FGF-9 groups. MI was induced by permanent coronary artery ligation and animals were subjected to 2D transthoracic echocardiography two weeks post-surgery. Immunohistochemical and immunoassay results from heart samples collected suggest significantly increased infiltration of monocytes (Mean ± SEM; MI: 2.02% ± 0.23% vs. Sham 0.75% ± 0.07%; p<0.05) and associated pro-inflammatory cytokines (TNF-α, MCP-1, and IL-6), adverse cardiac remodeling (Mean ± SEM; MI: 33% ± 3.04% vs. Sham 2.2% ± 0.33%; p<0.05), and left ventricular dysfunction (Mean ± SEM; MI: 35.4% ± 1.25% vs. Sham 49.19% ± 1.07%; p<0.05) in the MI group. Importantly, treatment of diabetic infarcted myocardium with FGF-9 resulted in significantly decreased monocyte infiltration (Mean ± SEM; MI+FGF-9: 1.39% ± 0.1% vs. MI: 2.02% ± 0.23%; p<0.05), increased M2 macrophage differentiation (Mean ± SEM; MI+FGF-9: 4.82% ± 0.86% vs. MI: 0.85% ± 0.3%; p<0.05) and associated anti-inflammatory cytokines (IL-10 and IL-1RA), reduced adverse remodeling (Mean ± SEM; MI+FGF-9: 11.59% ± 1.2% vs. MI: 33% ± 3.04%; p<0.05), and improved cardiac function (Fractional shortening, Mean ± SEM; MI+FGF-9: 41.51% ± 1.68% vs. MI: 35.4% ± 1.25%; p<0.05). In conclusion, our data suggest FGF-9 possesses novel therapeutic potential in its ability to

  2. Phage Particles as Vaccine Delivery Vehicles: Concepts, Applications and Prospects.

    PubMed

    Jafari, Narjes; Abediankenari, Saeid

    2015-01-01

    The development of new strategies for vaccine delivery for generating protective and long-lasting immune responses has become an expanding field of research. In the last years, it has been recognized that bacteriophages have several potential applications in the biotechnology and medical fields because of their intrinsic advantages, such as ease of manipulation and large-scale production. Over the past two decades, bacteriophages have gained special attention as vehicles for protein/peptide or DNA vaccine delivery. In fact, whole phage particles are used as vaccine delivery vehicles to achieve the aim of enhanced immunization. In this strategy, the carried vaccine is protected from environmental damage by phage particles. In this review, phage-based vaccine categories and their development are presented in detail, with discussion of the potential of phage-based vaccines for protection against microbial diseases and cancer treatment. Also reviewed are some recent advances in the field of phage- based vaccines.

  3. Phage Particles as Vaccine Delivery Vehicles: Concepts, Applications and Prospects.

    PubMed

    Jafari, Narjes; Abediankenari, Saeid

    2015-01-01

    The development of new strategies for vaccine delivery for generating protective and long-lasting immune responses has become an expanding field of research. In the last years, it has been recognized that bacteriophages have several potential applications in the biotechnology and medical fields because of their intrinsic advantages, such as ease of manipulation and large-scale production. Over the past two decades, bacteriophages have gained special attention as vehicles for protein/peptide or DNA vaccine delivery. In fact, whole phage particles are used as vaccine delivery vehicles to achieve the aim of enhanced immunization. In this strategy, the carried vaccine is protected from environmental damage by phage particles. In this review, phage-based vaccine categories and their development are presented in detail, with discussion of the potential of phage-based vaccines for protection against microbial diseases and cancer treatment. Also reviewed are some recent advances in the field of phage- based vaccines. PMID:26745034

  4. Exclusion of polyvalent T7-like phages by prophage elements.

    PubMed

    Faidiuk, I V; Tovkach, E I

    2014-01-01

    The study presents new insights into the process of interaction of T7-like bacteriophages FE44 and BA14 with lysogenic cells. It was demonstrated that single and double lysogens possess Abiphenotype regardless of genera, species and strain of bacteria that initially had normal phage sensitivity. Efficiency of plating of these phages is reduced by two orders of magnitude on monolysogens, whereas it decreases by 4-6 orders on bilysogens. In the latter case, phage infection leads to formation of more than 60% of aberrant capsids in phage progeny. Abortive phage infection is suggested to be associated with defects in general dynamics of the bacterial chromosome in single and double lysogens of Erwinia "horticola" and Escherichia coli. PMID:25434214

  5. Possible association between phages, Hoc protein, and the immune system.

    PubMed

    Dabrowska, K; Switała-Jeleń, K; Opolski, A; Górski, A

    2006-02-01

    Mammals have become "an environment" for enterobacterial phage life cycles. Therefore it could be expected that bacteriophages adapt to them. This adaptation must comprise bacteriophage proteins. Gp Hoc seems to have significance neither for phage particle structure nor for phage antibacterial activity. It is evidently not necessary for the "typical" antibacterial actions of bacteriophages. But the rules of evolution make it improbable that gp Hoc really has no function, and non-essential genes of T4-type phages are probably important for phages' adaptation to their particular lifestyle. More interesting is the eukaryotic origin of gp Hoc: a resemblance to immunoglobulin-like proteins that reflects their evolutionary relation. Substantial differences in biological activity between T4 and a mutant that lacks gp Hoc were observed in a mammalian system. Hoc protein seems to be one of the molecules predicted to interact with mammalian organisms and/or modulate these interactions. PMID:16195787

  6. Methods for identification of recombinants of phage lambda.

    PubMed

    Sanzey, B; Mercereau, O; Ternynck, T; Kourilsky, P

    1976-10-01

    Two methods are described which allow the screening of a large number of phage plaques for a specific DNA sequence carried by the phage or a specific antigen produced within the phage plaque. These methods were set up with lambda and lambdalac phages. Phage plaques were transferred onto nitrocellulose filters by desiccation in 0.1 M NaOH, and the lac sequence was detected by hybridization to radioactive lac mRNA. Beta-Galactosidase was detected by reaction with anti-beta-galactosidase immune serum included in the soft agar of the titration plates; the precipitate thus formed was revealed by means of enzyme-coupled antibodies and in situ coloration. These methods are potentially useful for the identification of lambda transducers, including those which are generated by in vitro recombination with eukaryotic DNA.

  7. Chemical strategies for the covalent modification of filamentous phage

    PubMed Central

    Bernard, Jenna M. L.; Francis, Matthew B.

    2014-01-01

    Historically filamentous bacteriophage have been known to be the workhorse of phage display due to their ability to link genotype to phenotype. More recently, the filamentous phage scaffold has proven to be powerful outside the realm of phage display technology in fields such as molecular imaging, cancer research and materials, and vaccine development. The ability of the virion to serve as a platform for a variety of applications heavily relies on the functionalization of the phage coat proteins with a wide variety of functionalities. Genetic modification of the coat proteins has been the most widely used strategy for functionalizing the virion; however, complementary chemical modification strategies can help to diversify the range of materials that can be developed. This review emphasizes the recent advances that have been made in the chemical modification of filamentous phage as well as some of the challenges that are involved in functionalizing the virion. PMID:25566240

  8. Nucleotide sequence of Bacillus phage Nf terminal protein gene.

    PubMed Central

    Leavitt, M C; Ito, J

    1987-01-01

    The nucleotide sequence of Bacillus phage Nf gene E has been determined. Gene E codes for phage terminal protein which is the primer necessary for the initiation of DNA replication. The deduced amino acid sequence of Nf terminal protein is approximately 66% homologous with the terminal proteins of Bacillus phages PZA and luminal diameter 29, and shows similar hydropathy and secondary structure predictions. A serine which has been identified as the residue which covalently links the protein to the 5' end of the genome in luminal diameter 29, is conserved in all three phages. The hydropathic and secondary structural environment of this serine is similar in these phage terminal proteins and also similar to the linking serine of adenovirus terminal protein. PMID:3601672

  9. Phage abortive infection in lactococci: variations on a theme.

    PubMed

    Chopin, Marie-Christine; Chopin, Alain; Bidnenko, Elena

    2005-08-01

    Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance.

  10. Filamentous Phages As a Model System in Soft Matter Physics.

    PubMed

    Dogic, Zvonimir

    2016-01-01

    Filamentous phages have unique physical properties, such as uniform particle lengths, that are not found in other model systems of rod-like colloidal particles. Consequently, suspensions of such phages provided powerful model systems that have advanced our understanding of soft matter physics in general and liquid crystals in particular. We described some of these advances. In particular we briefly summarize how suspensions of filamentous phages have provided valuable insight into the field of colloidal liquid crystals. We also describe recent experiments on filamentous phages that have elucidated a robust pathway for assembly of 2D membrane-like materials. Finally, we outline unique structural properties of filamentous phages that have so far remained largely unexplored yet have the potential to further advance soft matter physics and material science. PMID:27446051

  11. Filamentous Phages As a Model System in Soft Matter Physics.

    PubMed

    Dogic, Zvonimir

    2016-01-01

    Filamentous phages have unique physical properties, such as uniform particle lengths, that are not found in other model systems of rod-like colloidal particles. Consequently, suspensions of such phages provided powerful model systems that have advanced our understanding of soft matter physics in general and liquid crystals in particular. We described some of these advances. In particular we briefly summarize how suspensions of filamentous phages have provided valuable insight into the field of colloidal liquid crystals. We also describe recent experiments on filamentous phages that have elucidated a robust pathway for assembly of 2D membrane-like materials. Finally, we outline unique structural properties of filamentous phages that have so far remained largely unexplored yet have the potential to further advance soft matter physics and material science.

  12. Filamentous Phages As a Model System in Soft Matter Physics

    PubMed Central

    Dogic, Zvonimir

    2016-01-01

    Filamentous phages have unique physical properties, such as uniform particle lengths, that are not found in other model systems of rod-like colloidal particles. Consequently, suspensions of such phages provided powerful model systems that have advanced our understanding of soft matter physics in general and liquid crystals in particular. We described some of these advances. In particular we briefly summarize how suspensions of filamentous phages have provided valuable insight into the field of colloidal liquid crystals. We also describe recent experiments on filamentous phages that have elucidated a robust pathway for assembly of 2D membrane-like materials. Finally, we outline unique structural properties of filamentous phages that have so far remained largely unexplored yet have the potential to further advance soft matter physics and material science. PMID:27446051

  13. Transcriptomic and Metabolomic Analysis Revealed Multifaceted Effects of Phage Protein Gp70.1 on Pseudomonas aeruginosa

    PubMed Central

    Zhao, Xia; Chen, Canhuang; Jiang, Xingyu; Shen, Wei; Huang, Guangtao; Le, Shuai; Lu, Shuguang; Zou, Lingyun; Ni, Qingshan; Li, Ming; Zhao, Yan; Wang, Jing; Rao, Xiancai; Hu, Fuquan; Tan, Yinling

    2016-01-01

    The impact of phage infection on the host cell is severe. In order to take over the cellular machinery, some phage proteins were produced to shut off the host biosynthesis early in the phage infection. The discovery and identification of these phage-derived inhibitors have a significant prospect of application in antibacterial treatment. This work presented a phage protein, gp70.1, with non-specific inhibitory effects on Pseudomonas aeruginosa and Escherichia coli. Gp70.1 was encoded by early gene – orf 70.1 from P. aeruginosa phage PaP3. The P. aeruginosa with a plasmid encoding gp70.1 showed with delayed growth and had the appearance of a small colony. The combination of multifaceted analysis including microarray-based transcriptomic analysis, RT-qPCR, nuclear magnetic resonance (NMR) spectroscopy-based metabolomics and phenotype experiments were performed to investigate the effects of gp70.1 on P. aeruginosa. A total of 178 genes of P. aeruginosa mainly involved in extracellular function and metabolism were differentially expressed in the presence of gp70.1 at three examined time points. Furthermore, our results indicated that gp70.1 had an extensive impact on the extracellular phenotype of P. aeruginosa, such as motility, pyocyanin, extracellular protease, polysaccharide, and cellulase. For the metabolism of P. aeruginosa, the main effect of gp70.1 was the reduction of amino acid consumption. Finally, the RNA polymerase sigma factor RpoS was identified as a potential cellular target of gp70.1. Gp70.1 was the first bacterial inhibitor identified from Pseudomonas aeruginosa phage PaP3. It was also the first phage protein that interacted with the global regulator RpoS of bacteria. Our results indicated the potential value of gp70.1 in antibacterial applications. This study preliminarily revealed the biological function of gp70.1 and provided a reference for the study of other phage genes sharing similarities with orf70.1. PMID:27725812

  14. Challenges in predicting the evolutionary maintenance of a phage transgene

    PubMed Central

    2014-01-01

    Background In prior work, a phage engineered with a biofilm-degrading enzyme (dispersin B) cleared artificial, short-term biofilms more fully than the phage lacking the enzyme. An unresolved question is whether the transgene will be lost or maintained during phage growth – its loss would limit the utility of the engineering. Broadly supported evolutionary theory suggests that transgenes will be lost through a ‘tragedy of the commons’ mechanism unless the ecology of growth in biofilms meets specific requirements. We test that theory here. Results Functional properties of the transgenic phage were identified. Consistent with the previous study, the dispersin phage was superior to unmodified phage at clearing short term biofilms grown in broth, shown here to be an effect attributable to free enzyme. However, the dispersin phage was only marginally better than control phages on short term biofilms in minimal media and was no better than control phages in clearing long term biofilms. There was little empirical support for the tragedy of the commons framework despite a strong theoretical foundation for its supposed relevance. The framework requires that the transgene imposes an intrinsic cost, yet the transgene was intrinsically neutral or beneficial when expressed from one part of the phage genome. Expressed from a different part of the genome, the transgene did behave as if intrinsically costly, but its maintenance did not benefit from spatially structured growth per se – violating the tragedy framework. Conclusions Overall, the transgene was beneficial under many conditions, but no insight to its maintenance was attributable to the established evolutionary framework. The failure likely resides in system details that would be used to parameterize the models. Our study cautions against naive applications of evolutionary theory to synthetic biology, even qualitatively. PMID:25126112

  15. Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays

    PubMed Central

    Holguín, Angela V.; Rangel, Guillermo; Clavijo, Viviana; Prada, Catalina; Mantilla, Marcela; Gomez, María Catalina; Kutter, Elizabeth; Taylor, Corinda; Fineran, Peter C.; Barrios, Andrés Fernando González; Vives, Martha J.

    2015-01-01

    Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization. PMID:26274971

  16. Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays.

    PubMed

    Holguín, Angela V; Rangel, Guillermo; Clavijo, Viviana; Prada, Catalina; Mantilla, Marcela; Gomez, María Catalina; Kutter, Elizabeth; Taylor, Corinda; Fineran, Peter C; Barrios, Andrés Fernando González; Vives, Martha J

    2015-08-01

    Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization.

  17. Pretreatment with a 55-kDa tumor necrosis factor receptor-immunoglobulin fusion protein attenuates activation of coagulation, but not of fibrinolysis, during lethal bacteremia in baboons.

    PubMed

    van der Poll, T; Jansen, P M; Van Zee, K J; Hack, C E; Oldenburg, H A; Loetscher, H; Lesslauer, W; Lowry, S F; Moldawer, L L

    1997-07-01

    Baboons (Papio anubis) receiving a lethal intravenous infusion with live Escherichia coli were pretreated with either a 55-kDa tumor necrosis factor (TNF) receptor-IgG fusion protein (TNFR55:IgG) (n = 4, 4.6 mg/kg) or placebo (n = 4). Neutralization of TNF activity in TNFR55:IgG-treated animals was associated with a complete prevention of mortality and a strong attenuation of coagulation activation as reflected by the plasma concentrations of thrombin-antithrombin III complexes (P < .05). Activation of fibrinolysis was not influenced by TNFR55:IgG (plasma tissue-type plasminogen activator and plasmin-alpha2-antiplasmin complexes), whereas TNFR55:IgG did inhibit the release of plasminogen activator inhibitor type I (P < .05). Furthermore, TNFR55:IgG inhibited neutrophil degranulation (plasma levels of elastase-alpha1-antitrypsin complexes, P < .05) and modestly reduced release of secretory phospholipase A2. These data suggest that endogenous TNF contributes to activation of coagulation, but not to stimulation of fibrinolysis, during severe bacteremia.

  18. Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma-associated herpesvirus ORF57 protein is required for RNA splicing.

    PubMed

    Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan; Zheng, Zhi-Ming

    2014-11-01

    Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3-RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing.

  19. Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma–associated herpesvirus ORF57 protein is required for RNA splicing

    PubMed Central

    Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan

    2014-01-01

    Kaposi sarcoma–associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3–RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing. PMID:25234929

  20. An activator of protein kinase C (phorbol dibutyrate) attenuates atrial-natriuretic-factor-stimulated cyclic GMP accumulation in smooth-muscle cells.

    PubMed Central

    Nambi, P; Whitman, M; Aiyar, N; Stassen, F; Crooke, S T

    1987-01-01

    Rat thoracic aortic smooth-muscle cells (A-10; A.T.C.C. CRL 1476) displays a high density of vasopressin and atrial-natriuretic-factor (ANF) receptors and a low density of beta-adrenergic receptors. ANF stimulates cGMP (cyclic GMP) accumulation in a time- and dose-dependent fashion. Pretreatment of these cells with phorbol dibutyrate (PDBu), a known activator of protein kinase C, attenuated ANF-stimulated cGMP accumulation without affecting basal cGMP concentrations. This effect was concentration-dependent and was observed as early as 2 min after treatment. 4 alpha-Phorbol 12, 13-didecanoate (alpha PDD), which does not activate protein kinase C, did not inhibit the cGMP accumulation. PDBu pretreatment did not affect the density and affinity of ANF receptors. These data suggest that PDBu, presumably via activation of protein kinase C, might stimulate phosphorylation of a key regulatory protein in the ANF/cGMP pathway. PMID:2822009

  1. Involvement of leucine zipper transcription factor-like protein 1 (Lztfl1) in the attenuation of cognitive impairment by exercise training.

    PubMed

    Sakurai, Takuya; Ogasawara, Junetsu; Kizaki, Takako; Ishibashi, Yoshinaga; Fujiwara, Tomonori; Akagawa, Kimio; Izawa, Tetsuya; Oh-ishi, Shuji; Haga, Shukoh; Ohno, Hideki

    2011-12-01

    It is well known that exercise prevents and reduces cognitive impairment. In the present study, we focused on exercise training as a tool to prevent cognitive impairment, and searched for novel molecules that may relate to the prevention of cognitive impairment in the hippocampus. Two-month-old senescence-accelerated mouse prone-8 (SAMP8) mice were subjected to voluntary exercise training by running on a wheel for 4 months, and were then assigned a conditioned fear memory test. Moreover, various mRNA levels in the hippocampus were examined by DNA array analysis and real-time PCR. Contextual fear memory in SAMP8 control mice was significantly impaired compared with that in non-senescence mice. Exercise training definitely attenuated such cognitive impairment. The results of real-time PCR analysis that was conducted following DNA array analysis in the hippocampus revealed that, compared with SAMR8 control mice, the expression levels of leucine zipper transcription factor-like protein 1 (Lztfl1) mRNA were significantly higher in SAMP8 mice subjected to exercise training. In addition, the overexpression of Lztfl1 promoted neurite outgrowth in Neuro 2a cells. These results suggest that exercise has a preventive effect on cognitive impairment in SAMP8 mice, and that exercise-induced increase in Lztfl1 induces neurite outgrowth.

  2. The XXIIIrd Phage/Virus Assembly Meeting

    PubMed Central

    Serwer, Philip

    2014-01-01

    The XXIIIrd Phage/Virus Assembly (PVA) meeting returned to its birthplace in Lake Arrowhead, CA on September 8–13, 2013 (Fig. 1). The original meeting occurred in 1968, organized by Bob Edgar (Caltech, Pasadena, CA USA), Fred Eiserling (University of California, Los Angeles, Los Angeles, CA USA) and Bill Wood (Caltech, Pasadena, CA USA). The organizers of the 2013 meeting were Bill Gelbart (University of California, Los Angeles, Los Angeles, CA USA) and Jack Johnson (Scripps Research Institute, La Jolla, CA USA). This meeting specializes in an egalitarian format. Students are distinguished from senior faculty primarily by the signs of age. With the exception of historically based introductory talks, all talks were allotted the same time and freedom. This tradition began when the meeting was phage-only and has been continued now that all viruses are included. Many were the animated conversations about basic questions. New and international participants were present, a sign that the field has significant attraction, as it should, based on details below. The meeting was also characterized by a sense of humor and generally good times, a chance to both enjoy the science and forget the funding malaise to which many participants are exposed. I will present some of the meeting content, without attempting to be comprehensive. PMID:24498537

  3. Granulocyte-Macrophage Colony-Stimulating Factor Expressed by Recombinant Respiratory Syncytial Virus Attenuates Viral Replication and Increases the Level of Pulmonary Antigen-Presenting Cells

    PubMed Central

    Bukreyev, Alexander; Belyakov, Igor M.; Berzofsky, Jay A.; Murphy, Brian R.; Collins, Peter L.

    2001-01-01

    An obstacle to developing a vaccine against human respiratory syncytial virus (RSV) is that natural infection typically does not confer solid immunity to reinfection. To investigate methods to augment the immune response, recombinant RSV (rRSV) was constructed that expresses murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) from a transcription cassette inserted into the G-F intergenic region. Replication of rRSV/mGM-CSF in the upper and lower respiratory tracts of BALB/c mice was reduced 23- to 74- and 5- to 588-fold, respectively, compared to that of the parental rRSV. Despite this strong attenuation of replication, the level of RSV-specific serum antibodies induced by rRSV/mGM-CSF was comparable to, or marginally higher than, that of the parental rRSV. The induction of RSV-specific CD8+ cytotoxic T cells was moderately reduced during the initial infection, which might be a consequence of reduced antigen expression. Mice infected with rRSV/mGM-CSF had elevated levels of pulmonary mRNA for gamma interferon (IFN-γ) and interleukin 12 (IL-12) p40 compared to animals infected by wild-type rRSV. Elevated synthesis of IFN-γ could account for the restriction of RSV replication, as was observed previously with an IFN-γ-expressing rRSV. The accumulation of total pulmonary mononuclear cells and total CD4+ T lymphocytes was accelerated in animals infected with rRSV/mGM-CSF compared to that in animals infected with the control virus, and the level of IFN-γ-positive or IL-4-positive pulmonary CD4+ cells was elevated approximately twofold. The number of pulmonary lymphoid and myeloid dendritic cells and macrophages was increased up to fourfold in mice infected with rRSV/mGM-CSF compared to those infected with the parental rRSV, and the mean expression of major histocompatibility complex class II molecules, a marker of activation, was significantly increased in the two subsets of dendritic cells. Enhanced antigen presentation likely accounts for the

  4. Ligustrazine attenuates oxidative stress-induced activation of hepatic stellate cells by interrupting platelet-derived growth factor-β receptor-mediated ERK and p38 pathways

    SciTech Connect

    Zhang, Feng; Ni, Chunyan; Kong, Desong; Zhang, Xiaoping; Zhu, Xiaojing; Chen, Li; Lu, Yin; Zheng, Shizhong

    2012-11-15

    Hepatic fibrosis represents a frequent event following chronic insult to trigger wound healing reactions with accumulation of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is the pivotal event during liver fibrogenesis. Compelling evidence indicates that oxidative stress is concomitant with liver fibrosis irrespective of the underlying etiology. Natural antioxidant ligustrazine exhibits potent antifibrotic activities, but the mechanisms are poorly understood. Our studies were to investigate the ligustrazine effects on HSC activation stimulated by hydrogen peroxide (H{sub 2}O{sub 2}), an in vitro model mimicking the oxidative stress in liver fibrogenesis, and to elucidate the possible mechanisms. Our results demonstrated that H{sub 2}O{sub 2} at 5 μM significantly stimulated HSC proliferation and expression of marker genes of HSC activation; whereas ligustrazine dose-dependently suppressed proliferation and induced apoptosis in H{sub 2}O{sub 2}-activated HSCs, and attenuated expression of fibrotic marker genes. Mechanistic investigations revealed that ligustrazine reduced platelet-derived growth factor-β receptor (PDGF-βR) expression and blocked the phosphorylation of extracellular regulated protein kinase (ERK) and p38 kinase, two downstream effectors of PDGF-βR. Further molecular evidence suggested that ligustrazine interruption of ERK and p38 pathways was dependent on the blockade of PDGF-βR and might be involved in ligustrazine reduction of fibrotic marker gene expression under H{sub 2}O{sub 2} stimulation. Furthermore, ligustrazine modulated some proteins critical for HSC activation and ECM homeostasis in H{sub 2}O{sub 2}-stimulated HSCs. These data collectively indicated that ligustrazine could attenuate HSC activation caused by oxidative stress, providing novel insights into ligustrazine as a therapeutic option for hepatic fibrosis. Highlights: ► Ligustrazine inhibits oxidative stress-induced HSC activation.

  5. Luteolin is a bioflavonoid that attenuates adipocyte-derived inflammatory responses via suppression of nuclear factor-κB/mitogen-activated protein kinases pathway

    PubMed Central

    Nepali, Sarmila; Son, Ji-Seon; Poudel, Barun; Lee, Ji-Hyun; Lee, Young-Mi; Kim, Dae-Ki

    2015-01-01

    Background: Inflammation of adipocytes has been a therapeutic target for treatment of obesity and metabolic disorders which cause insulin resistance and hence lead to type II diabetes. Luteolin is a bioflavonoid with many beneficial properties such as antioxidant, antiproliferative, and anti-cancer. Objectives: To elucidate the potential anti-inflammatory response and the underlying mechanism of luteolin in 3T3-L1 adipocytes. Materials and Methods: We stimulated 3T3-L1 adipocytes with the mixture of tumor necrosis factor-α, lipopolysaccharide, and interferon-γ (TLI) in the presence or absence of luteolin. We performed Griess’ method for nitric oxide (NO) production and measure mRNA and protein expressions by real-time polymerase chain reaction and western blotting, respectively. Results: Luteolin opposed the stimulation of inducible nitric oxide synthase and NO production by simultaneous treatment of adipocytes with TLI. Furthermore, it reduced the pro-inflammatory genes such as cyclooxygenase-2, interleukin-6, resistin, and monocyte chemoattractant protein-1. Furthermore, luteolin improved the insulin sensitivity by enhancing the expression of insulin receptor substrates (IRS1/2) and glucose transporter-4 via phosphatidylinositol-3K signaling pathway. This inhibition was associated with suppression of Iκ-B-α degradation and subsequent inhibition of nuclear factor-κB (NF-κB) p65 translocation to the nucleus. In addition, luteolin blocked the phosphorylation of ERK1/2, c-Jun N-terminal Kinases and also p38 mitogen-activated protein kinases (MAPKs). Conclusions: These results illustrate that luteolin attenuates inflammatory responses in the adipocytes through suppression of NF-κB and MAPKs activation, and also improves insulin sensitivity in 3T3-L1 cells, suggesting that luteolin may represent a therapeutic agent to prevent obesity-associated inflammation and insulin resistance. PMID:26246742

  6. Associations of ghrelin with eating behaviors, stress, metabolic factors, and telomere length among overweight and obese women: Preliminary evidence of attenuated ghrelin effects in obesity?

    PubMed Central

    Buss, Julia; Havel, Peter J.; Epel, Elissa; Lin, Jue; Blackburn, Elizabeth; Daubenmier, Jennifer

    2014-01-01

    Ghrelin regulates homeostatic food intake, hedonic eating, and is a mediator in the stress response. In addition, ghrelin has metabolic, cardiovascular, and anti-aging effects. This cross-sectional study examined associations between total plasma ghrelin, caloric intake based on 3 day diet diaries, hedonic eating attitudes, stress-related and metabolic factors, and leukocyte telomere length in overweight (n=25) and obese women (n=22). We hypothesized associations between total plasma ghrelin and eating behaviors, stress, metabolic, cardiovascular, and cell aging factors among overweight women, but not among obese women due to lower circulating ghrelin levels and/or central resistance to ghrelin. Confirming previous studies demonstrating lowered plasma ghrelin in obesity, ghrelin levels were lower in the obese compared with overweight women. Among the overweight, ghrelin was positively correlated with caloric intake, giving in to cravings for highly palatable foods, and a flatter diurnal cortisol slope across 3 days. These relationships were non-significant among the obese group. Among overweight women, ghrelin was negatively correlated with insulin resistance, systolic blood pressure, and heart rate, and positively correlated with telomere length. Among the obese subjects, plasma ghrelin concentrations were negatively correlated with insulin resistance, but were not significantly correlated with blood pressure, heart rate or telomere length. Total plasma ghrelin and its associations with food intake, hedonic eating, and stress are decreased in obesity, providing evidence consistent with the theory that central resistance to ghrelin develops in obesity and ghrelin’s function in appetite regulation may have evolved to prevent starvation in food scarcity rather than cope with modern food excess. Furthermore, ghrelin is associated with metabolic and cardiovascular health, and may have anti-aging effects, but these effects may be attenuated in obesity. PMID:24462487

  7. Combined exercise and insulin-like growth factor-1 supplementation induces neurogenesis in old rats, but do not attenuate age-associated DNA damage.

    PubMed

    Koltai, Erika; Zhao, Zhongfu; Lacza, Zsombor; Cselenyak, Attila; Vacz, Gabriella; Nyakas, Csaba; Boldogh, Istvan; Ichinoseki-Sekine, Noriko; Radak, Zsolt

    2011-12-01

    We have investigated the effects of 2 weeks of insulin-like growth factor-1 (IGF-1) supplementation (5 μg/kg per day) and 6 weeks of exercise training (60% of the maximal oxygen consumption [VO₂ max]) on neurogenesis, DNA damage/repair, and sirtuin content in the hippocampus of young (3 months old) and old (26 months old) rats. Exercise improved the spatial memory of the old group, but IGF-1 supplementation eliminated this effect. An age-associated decrease in neurogenesis was attenuated by exercise and IGF-1 treatment. Aging increased the levels of 8-oxo-7,8-dihydroguanine (8-oxoG) and the protein Ku70, indicating the role of DNA damage in age-related neuropathology. Acetylation of 8-oxoguanine DNA glycosylase (OGG1) was detected in vivo, and this decreased with aging. However, in young animals, exercise and IGF-1 treatment increased acetylated (ac) OGG1 levels. Sirtuin 1 (SIRT1) and SIRT3, as DNA damage-associated lysine deacetylases, were measured, and SIRT1 decreased with aging, resulting in a large increase in acetylated lysine residues in the hippocampus. On the other hand, SIRT3 increased with aging. Exercise-induced neurogenesis might not be a causative factor of increased spatial memory, because IGF-1 plus exercise can induce neurogenesis in the hippocampus of older rats. Data revealed that the age-associated increase in 8-oxoG levels is due to decreased acetylation of OGG1. Age-associated decreases in SIRT1 and the associated increase in lysine acetylation, in the hippocampus, could have significant impact on function and thus, could suggest a therapeutic target.

  8. Associations of ghrelin with eating behaviors, stress, metabolic factors, and telomere length among overweight and obese women: preliminary evidence of attenuated ghrelin effects in obesity?

    PubMed

    Buss, Julia; Havel, Peter J; Epel, Elissa; Lin, Jue; Blackburn, Elizabeth; Daubenmier, Jennifer

    2014-05-01

    Ghrelin regulates homeostatic food intake, hedonic eating, and is a mediator in the stress response. In addition, ghrelin has metabolic, cardiovascular, and anti-aging effects. This cross-sectional study examined associations between total plasma ghrelin, caloric intake based on 3day diet diaries, hedonic eating attitudes, stress-related and metabolic factors, and leukocyte telomere length in overweight (n=25) and obese women (n=22). We hypothesized associations between total plasma ghrelin and eating behaviors, stress, metabolic, cardiovascular, and cell aging factors among overweight women, but not among obese women due to lower circulating ghrelin levels and/or central resistance to ghrelin. Confirming previous studies demonstrating lowered plasma ghrelin in obesity, ghrelin levels were lower in the obese compared with overweight women. Among the overweight, ghrelin was positively correlated with caloric intake, giving in to cravings for highly palatable foods, and a flatter diurnal cortisol slope across 3days. These relationships were non-significant among the obese group. Among overweight women, ghrelin was negatively correlated with insulin resistance, systolic blood pressure, and heart rate, and positively correlated with telomere length. Among the obese subjects, plasma ghrelin concentrations were negatively correlated with insulin resistance, but were not significantly correlated with blood pressure, heart rate or telomere length. Total plasma ghrelin and its associations with food intake, hedonic eating, and stress are decreased in obesity, providing evidence consistent with the theory that central resistance to ghrelin develops in obesity and ghrelin's function in appetite regulation may have evolved to prevent starvation in food scarcity rather than cope with modern food excess. Furthermore, ghrelin is associated with metabolic and cardiovascular health, and may have anti-aging effects, but these effects may be attenuated in obesity.

  9. Nuclear factor erythroid 2-related factor 2 antibody attenuates thermal hyperalgesia in the dorsal root ganglion: Neurochemical changes and behavioral studies after sciatic nerve-pinch injury.

    PubMed

    Xiang, Qiong; Yu, Chao; Zhu, Yao-Feng; Li, Chun-Yan; Tian, Rong-Bo; Li, Xian-Hui

    2016-08-01

    Oxidative stress is generated in several peripheral nerve injury models.Nuclear factor erythroid 2-related factor 2 (Nrf2) is activated to have a role in antioxidant effect. After nerve injury, the severely painful behavior is also performed. However, little has been explored regarding the function of Nrf2 in this painful process. Therefore, in this study, we compared the effects of Nrf2 antibody administration following sciatic nerve-pinch injury on painful behavior induced in young mice and neurochemical changes in dorsal root ganglion neurons. After pinch nerve injury, we found that the magnitude of the thermal allodynia was significantly decreased after application of Nrf2 antibody (5ul, 1mg/ml) in such injured animals and phosphorylated ERK(p-ERK) as well as the apoptotic protein (i.e., Bcl-6) in DRG neurons were also down-regulated in the anti-Nrf2-treated injured groups compared to the saline-treated groups. Taken collectively, these data suggested that the Nrf2 antibody reduced thermal hyperalgesia via ERK pathway and the down regulation of Bcl-6 protein from the apoptosis pathway might be protecting against the protein deletions caused by anti-Nrf2 effect and suggested the new therapeutic strategy with Nrf2 inhibitor following nerve injury. PMID:27316447

  10. Biofilm control with natural and genetically-modified phages.

    PubMed

    Motlagh, Amir Mohaghegh; Bhattacharjee, Ananda Shankar; Goel, Ramesh

    2016-04-01

    Bacteriophages, as the most dominant and diverse entities in the universe, have the potential to be one of the most promising therapeutic agents. The emergence of multidrug-resistant bacteria and the antibiotic crisis in the last few decades have resulted in a renewed interest in phage therapy. Furthermore, bacteriophages, with the capacity to rapidly infect and overcome bacterial resistance, have demonstrated a sustainable approach against bacterial pathogens-particularly in biofilm. Biofilm, as complex microbial communities located at interphases embedded in a matrix of bacterial extracellular polysaccharide substances (EPS), is involved in health issues such as infections associated with the use of biomaterials and chronic infections by multidrug resistant bacteria, as well as industrial issues such as biofilm formation on stainless steel surfaces in food industry and membrane biofouling in water and wastewater treatment processes. In this paper, the most recent studies on the potential of phage therapy using natural and genetically-modified lytic phages and their associated enzymes in fighting biofilm development in various fields including engineering, industry, and medical applications are reviewed. Phage-mediated prevention approaches as an indirect phage therapy strategy are also explored in this review. In addition, the limitations of these approaches and suggestions to overcome these constraints are discussed to enhance the efficiency of phage therapy process. Finally, future perspectives and directions for further research towards a better understanding of phage therapy to control biofilm are recommended.

  11. Coevolution of CRISPR bacteria and phage in 2 dimensions

    NASA Astrophysics Data System (ADS)

    Han, Pu; Deem, Michael

    2014-03-01

    CRISPR (cluster regularly interspaced short palindromic repeats) is a newly discovered adaptive, heritable immune system of prokaryotes. It can prevent infection of prokaryotes by phage. Most bacteria and almost all archae have CRISPR. The CRISPR system incorporates short nucleotide sequences from viruses. These incorporated sequences provide a historical record of the host and predator coevolution. We simulate the coevolution of bacteria and phage in 2 dimensions. Each phage has multiple proto-spacers that the bacteria can incorporate. Each bacterium can store multiple spacers in its CRISPR. Phages can escape recognition by the CRISPR system via point mutation or recombination. We will discuss the different evolutionary consequences of point mutation or recombination on the coevolution of bacteria and phage. We will also discuss an intriguing ``dynamic phase transition'' in the number of phage as a function of time and mutation rate. We will show that due to the arm race between phages and bacteria, the frequency of spacers and proto-spacers in a population can oscillate quite rapidly.

  12. Phage Therapy – Everything Old is New Again

    PubMed Central

    Kropinski, Andrew M

    2006-01-01

    The study of bacterial viruses (bacteriophages or phages) proved pivotal in the nascence of the disciplines of molecular biology and microbial genetics, providing important information on the central processes of the bacterial cell (DNA replication, transcription and translation) and on how DNA can be transferred from one cell to another. As a result of the pioneering genetics studies and modern genomics, it is now known that phages have contributed to the evolution of the microbial cell and to its pathogenic potential. Because of their ability to transmit genes, phages have been exploited to develop cloning vector systems. They also provide a plethora of enzymes for the modern molecular biologist. Until the introduction of antibiotics, phages were used to treat bacterial infections (with variable success). Western science is now having to re-evaluate the application of phage therapy – a therapeutic modality that never went out of vogue in Eastern Europe – because of the emergence of an alarming number of antibiotic-resistant bacteria. The present article introduces the reader to phage biology, and the benefits and pitfalls of phage therapy in humans and animals. PMID:18382643

  13. Tumor cell-targeting by phage-displayed peptides.

    PubMed

    Rasmussen, Ulla B; Schreiber, Valerie; Schultz, Huguette; Mischler, Fabienne; Schughart, Klaus

    2002-07-01

    We isolated cancer cell-specific phages by subtracting and selecting complex peptide display phage libraries on cultured human cancer cells. The best candidate was selected by performing three rounds of subtraction before each of five selections on the human colorectal WiDr cell line. The phage showed more than 1000-fold higher binding efficiency for WiDr cells when compared to five other human cancer cell lines, including two of colorectal origin, and when compared to wild-type M13 phage. Fifty-fold higher binding efficiency was also seen for a human breast cancer cell line. We show that the WiDr cell binding of the selected phage was efficiently competed by the synthetic peptide HEWSYLAPYPWF, predicted from the phage sequence. This confirms that the specificity of the peptide is independent of the display by the phage coat proteins. The identified peptide may target biomarkers linked to colorectal cancer, and thus be useful for designing gene transfer vectors as well as diagnostic and prognostic tools for this disease. PMID:12082461

  14. Chemical and Genetic Wrappers for Improved Phage and RNA Display

    PubMed Central

    Lamboy, Jorge A.; Tam, Phillip Y.; Lee, Lucie S.; Jackson, Pilgrim J.; Avrantinis, Sara K.; Lee, Hye Jin; Corn, Robert M.

    2009-01-01

    An Achilles heel inherent to all molecular display formats, background binding between target and display system introduces false positives into screens and selections. For example, the negatively charged surfaces of phage, mRNA, and ribosome display systems bind with unacceptably high non-specificity to positively charged target molecules, which represent an estimated 35% of proteins in the human proteome. We report the first systematic attempt to understand why a broad class of molecular display selections fail, and then solve the underlying problem for both phage and RNA display. First, a genetic strategy introduced a short charge neutralizing peptide into the solvent-exposed, negatively charged phage coat. The modified phage (KO7+) reduced or eliminated non-specific binding to the problematic high pI proteins. In the second, chemical approach, oligolysine wrappers for phage and total RNA blocked non-specific interactions. For phage display applications, the peptides Lysn (where n = 16 to 24) emerged as optimal for wrapping the phage. Lys8, however, provided effective wrappers for RNA binding in assays against the RNA binding protein HIV-1 Vif. The oligolysine peptides blocked non-specific binding to allow successful selections, screens, and assays with five previously unworkable protein targets. PMID:18973165

  15. Phage-bacteria infection networks: From nestedness to modularity

    NASA Astrophysics Data System (ADS)

    Flores, Cesar O.; Valverde, Sergi; Weitz, Joshua S.

    2013-03-01

    Bacteriophages (viruses that infect bacteria) are the most abundant biological life-forms on Earth. However, very little is known regarding the structure of phage-bacteria infections. In a recent study we re-evaluated 38 prior studies and demonstrated that phage-bacteria infection networks tend to be statistically nested in small scale communities (Flores et al 2011). Nestedness is consistent with a hierarchy of infection and resistance within phages and bacteria, respectively. However, we predicted that at large scales, phage-bacteria infection networks should be typified by a modular structure. We evaluate and confirm this hypothesis using the most extensive study of phage-bacteria infections (Moebus and Nattkemper 1981). In this study, cross-infections were evaluated between 215 marine phages and 286 marine bacteria. We develop a novel multi-scale network analysis and find that the Moebus and Nattkemper (1981) study, is highly modular (at the whole network scale), yet also exhibits nestedness and modularity at the within-module scale. We examine the role of geography in driving these modular patterns and find evidence that phage-bacteria interactions can exhibit strong similarity despite large distances between sites. CFG acknowledges the support of CONACyT Foundation. JSW holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund and acknowledges the support of the James S. McDonnell Foundation

  16. Biofilm control with natural and genetically-modified phages.

    PubMed

    Motlagh, Amir Mohaghegh; Bhattacharjee, Ananda Shankar; Goel, Ramesh

    2016-04-01

    Bacteriophages, as the most dominant and diverse entities in the universe, have the potential to be one of the most promising therapeutic agents. The emergence of multidrug-resistant bacteria and the antibiotic crisis in the last few decades have resulted in a renewed interest in phage therapy. Furthermore, bacteriophages, with the capacity to rapidly infect and overcome bacterial resistance, have demonstrated a sustainable approach against bacterial pathogens-particularly in biofilm. Biofilm, as complex microbial communities located at interphases embedded in a matrix of bacterial extracellular polysaccharide substances (EPS), is involved in health issues such as infections associated with the use of biomaterials and chronic infections by multidrug resistant bacteria, as well as industrial issues such as biofilm formation on stainless steel surfaces in food industry and membrane biofouling in water and wastewater treatment processes. In this paper, the most recent studies on the potential of phage therapy using natural and genetically-modified lytic phages and their associated enzymes in fighting biofilm development in various fields including engineering, industry, and medical applications are reviewed. Phage-mediated prevention approaches as an indirect phage therapy strategy are also explored in this review. In addition, the limitations of these approaches and suggestions to overcome these constraints are discussed to enhance the efficiency of phage therapy process. Finally, future perspectives and directions for further research towards a better understanding of phage therapy to control biofilm are recommended. PMID:26931607

  17. Review: phage therapy: a modern tool to control bacterial infections.

    PubMed

    Qadir, Muhammad Imran

    2015-01-01

    The evolution of antibiotic-resistant in bacteria has aggravated curiosity in development of alternative therapy to conventional drugs. One of the emerging drugs that can be used alternative to antibiotics is bacteriophage therapy. The use of living phages in the cure of lethal infectious life threatening diseases caused by Gram positive and Gram negative bacteria has been reported. Another development in the field of bacteriophage therapy is the use of genetically modified and non replicating phages in the treatment of bacterial infection. Genetically engineered bacteriophages can be used as adjuvant along with antibiotic therapy. Phages encoded with lysosomal enzymes are also effectual in the treatment of infectious diseases.

  18. The Resistance of Vibrio cholerae O1 El Tor Strains to the Typing Phage 919TP, a Member of K139 Phage Family.

    PubMed

    Shen, Xiaona; Zhang, Jingyun; Xu, Jialiang; Du, Pengcheng; Pang, Bo; Li, Jie; Kan, Biao

    2016-01-01

    Bacteriophage 919TP is a temperate phage of Vibrio cholerae serogroup O1 El Tor and is used as a subtyping phage in the phage-biotyping scheme in cholera surveillance in China. In this study, sequencing of the 919TP genome showed that it belonged to the Vibrio phage K139 family. The mechanisms conferring resistance to 919TP infection of El Tor strains were explored to help understand the subtyping basis of phage 919TP and mutations related to 919TP resistance. Among the test strains resistant to phage 919TP, most contained the temperate 919TP phage genome, which facilitated superinfection exclusion to 919TP. Our data suggested that this immunity to Vibrio phage 919TP occurred after absorption of the phage onto the bacteria. Other strains contained LPS receptor synthesis gene mutations that disable adsorption of phage 919TP. Several strains resistant to 919TP infection possessed unknown resistance mechanisms, since they did not contain LPS receptor mutations or temperate K139 phage genome. Further research is required to elucidate the phage infection steps involved in the resistance of these strains to phage infection. PMID:27242744

  19. The Resistance of Vibrio cholerae O1 El Tor Strains to the Typing Phage 919TP, a Member of K139 Phage Family

    PubMed Central

    Shen, Xiaona; Zhang, Jingyun; Xu, Jialiang; Du, Pengcheng; Pang, Bo; Li, Jie; Kan, Biao

    2016-01-01

    Bacteriophage 919TP is a temperate phage of Vibrio cholerae serogroup O1 El Tor and is used as a subtyping phage in the phage-biotyping scheme in cholera surveillance in China. In this study, sequencing of the 919TP genome showed that it belonged to the Vibrio phage K139 family. The mechanisms conferring resistance to 919TP infection of El Tor strains were explored to help understand the subtyping basis of phage 919TP and mutations related to 919TP resistance. Among the test strains resistant to phage 919TP, most contained the temperate 919TP phage genome, which facilitated superinfection exclusion to 919TP. Our data suggested that this immunity to Vibrio phage 919TP occurred after absorption of the phage onto the bacteria. Other strains contained LPS receptor synthesis gene mutations that disable adsorption of phage 919TP. Several strains resistant to 919TP infection possessed unknown resistance mechanisms, since they did not contain LPS receptor mutations or temperate K139 phage genome. Further research is required to elucidate the phage infection steps involved in the resistance of these strains to phage infection. PMID:27242744

  20. Inhibition of Plasminogen Activator Inhibitor-1 Attenuates Transforming Growth Factor-β-Dependent Epithelial Mesenchymal Transition and Differentiation of Fibroblasts to Myofibroblasts

    PubMed Central

    Omori, Keitaro; Hattori, Noboru; Senoo, Tadashi; Takayama, Yusuke; Masuda, Takeshi; Nakashima, Taku; Iwamoto, Hiroshi; Fujitaka, Kazunori; Hamada, Hironobu; Kohno, Nobuoki

    2016-01-01

    Transforming growth factor-β (TGF-β) is central during the pathogenesis of pulmonary fibrosis, in which the plasminogen activator inhibitor-1 (PAI-1) also has an established role. TGF-β is also known to be the strongest inducer of PAI-1. To investigate the link between PAI-1 and TGF-β in fibrotic processes, we evaluated the effect of SK-216, a PAI-1-specific inhibitor, in TGF-β-dependent epithelial-mesenchymal transition (EMT) and fibroblast to myofibroblast differentiation. In human alveolar epithelial A549 cells, treatment with TGF-β induced EMT, whereas co-treatment with SK-216 attenuated the occurrence of EMT. The inhibition of TGF-β-induced EMT by SK-216 was also confirmed in the experiment using murine epithelial LA-4 cells. Blocking EMT by SK-216 inhibited TGF-β-induced endogenous production of PAI-1 and TGF-β in A549 cells as well. These effects of SK-216 were not likely mediated by suppressing either Smad or ERK pathways. Using human lung fibroblast MRC-5 cells, we demonstrated that SK-216 inhibited TGF-β-dependent differentiation of fibroblasts to myofibroblasts. We also observed this inhibition by SK-216 in human primary lung fibroblasts. Following these in vitro results, we tested oral administration of SK-216 into mice injected intratracheally with bleomycin. We found that SK-216 reduced the degree of bleomycin-induced pulmonary fibrosis in mice. Although the precise mechanisms underlying the link between TGF-β and PAI-1 regarding fibrotic process were not determined, PAI-1 seems to act as a potent downstream effector on the pro-fibrotic property of TGF-β. In addition, inhibition of PAI-1 activity by a PAI-1 inhibitor exerts an antifibrotic effect even in vivo. These data suggest that targeting PAI-1 as a downstream effector of TGF-β could be a promising therapeutic strategy for pulmonary fibrosis. PMID:26859294

  1. Inhibition of Plasminogen Activator Inhibitor-1 Attenuates Transforming Growth Factor-β-Dependent Epithelial Mesenchymal Transition and Differentiation of Fibroblasts to Myofibroblasts.

    PubMed

    Omori, Keitaro; Hattori, Noboru; Senoo, Tadashi; Takayama, Yusuke; Masuda, Takeshi; Nakashima, Taku; Iwamoto, Hiroshi; Fujitaka, Kazunori; Hamada, Hironobu; Kohno, Nobuoki

    2016-01-01

    Transforming growth factor-β (TGF-β) is central during the pathogenesis of pulmonary fibrosis, in which the plasminogen activator inhibitor-1 (PAI-1) also has an established role. TGF-β is also known to be the strongest inducer of PAI-1. To investigate the link between PAI-1 and TGF-β in fibrotic processes, we evaluated the effect of SK-216, a PAI-1-specific inhibitor, in TGF-β-dependent epithelial-mesenchymal transition (EMT) and fibroblast to myofibroblast differentiation. In human alveolar epithelial A549 cells, treatment with TGF-β induced EMT, whereas co-treatment with SK-216 attenuated the occurrence of EMT. The inhibition of TGF-β-induced EMT by SK-216 was also confirmed in the experiment using murine epithelial LA-4 cells. Blocking EMT by SK-216 inhibited TGF-β-induced endogenous production of PAI-1 and TGF-β in A549 cells as well. These effects of SK-216 were not likely mediated by suppressing either Smad or ERK pathways. Using human lung fibroblast MRC-5 cells, we demonstrated that SK-216 inhibited TGF-β-dependent differentiation of fibroblasts to myofibroblasts. We also observed this inhibition by SK-216 in human primary lung fibroblasts. Following these in vitro results, we tested oral administration of SK-216 into mice injected intratracheally with bleomycin. We found that SK-216 reduced the degree of bleomycin-induced pulmonary fibrosis in mice. Although the precise mechanisms underlying the link between TGF-β and PAI-1 regarding fibrotic process were not determined, PAI-1 seems to act as a potent downstream effector on the pro-fibrotic property of TGF-β. In addition, inhibition of PAI-1 activity by a PAI-1 inhibitor exerts an antifibrotic effect even in vivo. These data suggest that targeting PAI-1 as a downstream effector of TGF-β could be a promising therapeutic strategy for pulmonary fibrosis.

  2. Sodium nitrite attenuates hypertension-in-pregnancy and blunts increases in soluble fms-like tyrosine kinase-1 and in vascular endothelial growth factor.

    PubMed

    Gonçalves-Rizzi, Victor Hugo; Possomato-Vieira, Jose Sergio; Sales Graça, Tamiris Uracs; Nascimento, Regina Aparecida; Dias-Junior, Carlos A

    2016-07-01

    Preeclampsia is a pregnancy-associated disorder characterized by hypertension with uncertain pathogenesis. Increases in antiangiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) and reductions in nitric oxide (NO) bioavailability have been observed in preeclamptic women. However, the specific mechanisms linking these detrimental changes to the hypertension-in-pregnancy are not clearly understood. In this regard, while recent findings have suggested that nitrite-derived NO formation exerts antihypertensive and antioxidant effects, no previous study has examined these responses to orally administered nitrite in hypertension-in-pregnancy. We then hypothesized restoring NO bioavailability with sodium nitrite in pregnant rats upon NO synthesis inhibition with N(omega)-nitro-l-arginine methyl ester (L-NAME) attenuates hypertension and high circulating levels of sFlt-1. Number and weight of pups and placentae were recorded to assess maternal-fetal interface. Plasma sFlt-1, vascular endothelial growth factor (VEGF) and biochemical determinants of NO formation and of antioxidant function were measured. We found that sodium nitrite blunts the hypertension-in-pregnancy and restores the NO bioavailability, and concomitantly prevents the L-NAME-induced high circulating sFlt-1 and VEGF levels. Also, our results suggest that nitrite-derived NO protected against reductions in litter size and placental weight caused by L-NAME, improving number of viable and resorbed fetuses and antioxidant function. Therefore, the present findings are consistent with the hypothesis that nitrite-derived NO may possibly be the driving force behind the maternal and fetal beneficial effects observed with sodium nitrite during hypertension-in-pregnancy. Certainly further investigations are required in preeclampsia, since counteracting the damages to the mother and fetal sides resulting from hypertension and elevated sFlt-1 levels may provide a great benefit in this gestational hypertensive disease

  3. Donepezil, an acetylcholinesterase inhibitor, attenuates LPS-induced inflammatory response in murine macrophage cell line RAW 264.7 through inhibition of nuclear factor kappa B translocation.

    PubMed

    Arikawa, Mikihiko; Kakinuma, Yoshihiko; Noguchi, Tatsuya; Todaka, Hiroshi; Sato, Takayuki

    2016-10-15

    We have previously demonstrated that the pharmacotherapy with donepezil, an acetylcholinesterase inhibitor, suppresses cardiac remodeling in a mouse model of ischemic heart failure after myocardial infarction (MI). However, the precise mechanisms of the cardioprotective effect of donepezil have not been completely delineated. Because post-ischemic inflammation is a pathological key event in the cardiac remodeling process following MI, we investigated the hypothesis that donepezil acts as an inhibitor of inflammatory mediators. RAW 264.7 murine macrophage cells were pretreated with donepezil (100µM) prior to a pro-inflammatory stimulation by administration of lipopolysaccharide (LPS, 10ng/ml). Donepezil significantly reduced intra- and extracellular levels of various kinds of inflammatory mediators such as TNF-α, IL-1β, IL-2, IL-6 and IL-18 after the LPS stimulation, and attenuated LPS-induced nuclear translocation of nuclear factor-kappa B (NF-κB). These results indicate that donepezil possesses an anti-inflammatory property. However, the inhibitory effect of donepezil on the macrophage inflammatory responses was never reproduced by ACh, nor was disrupted by ACh receptor blockers. Moreover, other kinds of acetylcholinesterase inhibitors failed to inhibit the inflammatory responses in LPS-stimulated macrophage cells. These results suggest that a cholinergic anti-inflammatory pathway would not be involved in the anti-inflammatory effect of donepezil and that the specific characteristics of donepezil in suppressing the LPS-induced cytokine release and the NF-κB activation would be independent of its acetylcholinesterase inhibition. The present study showed that donepezil exerts an anti-inflammatory effect independently of acetylcholinesterase inhibitory action, thereby donepezil may contribute to cardioprotection during cardiac remodeling process in an ischemic heart failure after MI.

  4. Inhibition of corticotropin releasing factor expression in the central nucleus of the amygdala attenuates stress-induced behavioral and endocrine responses

    PubMed Central

    Callahan, Leah B.; Tschetter, Kristi E.; Ronan, Patrick J.

    2013-01-01

    Corticotropin releasing factor (CRF) is a primary mediator of endocrine, autonomic and behavioral stress responses. Studies in both humans and animal models have implicated CRF in a wide-variety of psychiatric conditions including anxiety disorders such as post-traumatic stress disorder (PTSD), depression, sleep disorders and addiction among others. The central nucleus of the amygdala (CeA), a key limbic structure with one of the highest concentrations of CRF-producing cells outside of the hypothalamus, has been implicated in anxiety-like behavior and a number of stress-induced disorders. This study investigated the specific role of CRF in the CeA on both endocrine and behavioral responses to stress. We used RNA Interference (RNAi) techniques to locally and specifically knockdown CRF expression in CeA. Behavior was assessed using the elevated plus maze (EPM) and open field test (OF). Knocking down CRF expression in the CeA had no significant effect on measures of anxiety-like behavior in these tests. However, it did have an effect on grooming behavior, a CRF-induced behavior. Prior exposure to a stressor sensitized an amygdalar CRF effect on stress-induced HPA activation. In these stress-challenged animals silencing CRF in the CeA significantly attenuated corticosterone responses to a subsequent behavioral stressor. Thus, it appears that while CRF projecting from the CeA does not play a significant role in the expression stress-induced anxiety-like behaviors on the EPM and OF it does play a critical role in stress-induced HPA activation. PMID:24194694

  5. Inactivation of Burkholderia cepacia Complex Phage KS9 gp41 Identifies the Phage Repressor and Generates Lytic Virions▿ †

    PubMed Central

    Lynch, Karlene H.; Seed, Kimberley D.; Stothard, Paul; Dennis, Jonathan J.

    2010-01-01

    The Burkholderia cepacia complex (BCC) is made up of at least 17 species of Gram-negative opportunistic bacterial pathogens that cause fatal infections in patients with cystic fibrosis and chronic granulomatous disease. KS9 (vB_BcenS_KS9), one of a number of temperate phages isolated from BCC species, is a prophage of Burkholderia pyrrocinia LMG 21824. Transmission electron micrographs indicate that KS9 belongs to the family Siphoviridae and exhibits the B1 morphotype. The 39,896-bp KS9 genome, comprised of 50 predicted genes, integrates into the 3′ end of the LMG 21824 GTP cyclohydrolase II open reading frame. The KS9 genome is most similar to uncharacterized prophage elements in the genome of B. cenocepacia PC184 (vB_BcenZ_ PC184), as well as Burkholderia thailandensis phage φE125 and Burkholderia pseudomallei phage φ1026b. Using molecular techniques, we have disrupted KS9 gene 41, which exhibits similarity to genes encoding phage repressors, producing a lytic mutant named KS9c. This phage is incapable of stable lysogeny in either LMG 21824 or B. cenocepacia strain K56-2 and rescues a Galleria mellonella infection model from experimental B. cenocepacia K56-2 infections at relatively low multiplicities of infection. These results readily demonstrate that temperate phages can be genetically engineered to lytic form and that these modified phages can be used to treat bacterial infections in vivo. PMID:19939932

  6. Characterization of novel phages isolated in coagulase-negative staphylococci reveals evolutionary relationships with Staphylococcus aureus phages.

    PubMed

    Deghorain, Marie; Bobay, Louis-Marie; Smeesters, Pierre R; Bousbata, Sabrina; Vermeersch, Marjorie; Perez-Morga, David; Drèze, Pierre-Alexandre; Rocha, Eduardo P C; Touchon, Marie; Van Melderen, Laurence

    2012-11-01

    Despite increasing interest in coagulase-negative staphylococci (CoNS), little information is available about their bacteriophages. We isolated and sequenced three novel temperate Siphoviridae phages (StB12, StB27, and StB20) from the CoNS Staphylococcus hominis and S. capitis species. The genome sizes are around 40 kb, and open reading frames (ORFs) are arranged in functional modules encoding lysogeny, DNA metabolism, morphology, and cell lysis. Bioinformatics analysis allowed us to assign a potential function to half of the predicted proteins. Structural elements were further identified by proteomic analysis of phage particles, and DNA-packaging mechanisms were determined. Interestingly, the three phages show identical integration sites within their host genomes. In addition to this experimental characterization, we propose a novel classification based on the analysis of 85 phage and prophage genomes, including 15 originating from CoNS. Our analysis established 9 distinct clusters and revealed close relationships between S. aureus and CoNS phages. Genes involved in DNA metabolism and lysis and potentially in phage-host interaction appear to be widespread, while structural genes tend to be cluster specific. Our findings support the notion of a possible reciprocal exchange of genes between phages originating from S. aureus and CoNS, which may be of crucial importance for pathogenesis in staphylococci.

  7. CRISPR/Cas9-mediated phage resistance is not impeded by the DNA modifications of phage T4.

    PubMed

    Yaung, Stephanie J; Esvelt, Kevin M; Church, George M

    2014-01-01

    Bacteria rely on two known DNA-level defenses against their bacteriophage predators: restriction-modification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems. Certain phages have evolved countermeasures that are known to block endonucleases. For example, phage T4 not only adds hydroxymethyl groups to all of its cytosines, but also glucosylates them, a strategy that defeats almost all restriction enzymes. We sought to determine whether these DNA modifications can similarly impede CRISPR-based defenses. In a bioinformatics search, we found naturally occurring CRISPR spacers that potentially target phages known to modify their DNA. Experimentally, we show that the Cas9 nuclease from the Type II CRISPR system of Streptococcus pyogenes can overcome a variety of DNA modifications in Escherichia coli. The levels of Cas9-mediated phage resistance to bacteriophage T4 and the mutant phage T4 gt, which contains hydroxymethylated but not glucosylated cytosines, were comparable to phages with unmodified cytosines, T7 and the T4-like phage RB49. Our results demonstrate that Cas9 is not impeded by N6-methyladenine, 5-methylcytosine, 5-hydroxymethylated cytosine, or glucosylated 5-hydroxymethylated cytosine.

  8. Hydroxyapatite chromatography of phage-display virions.

    PubMed

    Smith, George P; Gingrich, Todd R

    2005-12-01

    Hydroxyapatite column chromatography can be used to purify filamentous bacteriophage--the phage most commonly used for phage display. Virions that have been partially purified from culture supernatant by two cycles of precipitation in 2% polyethylene glycol are adsorbed onto the matrix at a density of at least 7.6 x 10(13) virions (about 3 mg) per milliliter of packed bed volume in phosphate-buffered saline (PBS; 0.15 M NaCl, 5 mM NaH2PO4, pH-adjusted to 7.0 with NaOH). The matrix is washed successively with wash buffer I(150 mM NaCl, 125 mM phosphate, pH 7.0), wash buffer II (2.55 M NaCl, 125 mM phosphate, pH 7.0), and wash buffer I; after which virions are desorbed in desorption buffer (150 mM NaCl, 200 mM phosphate, pH 7.0), and the matrix is stripped with stripping buffer (150 mM NaCl, 1 Mphosphate, pH 7.0). About half of the applied virions are recovered in desorption buffer. Western blot analysis shows that they have undetectable levels of host-derived protein contaminants that are present in the input virions and in virions purified by CsCl equilibrium density gradient centrifugation--the method most commonly used to prepare virions in high purity. Hydroxyapatite chromatography is thus an attractive alternative method for purifying filamentous virions, particularly when the scale is too large for ultracentrifugation to be practical. PMID:16382907

  9. DC attenuation meter

    DOEpatents

    Hargrove, Douglas L.

    2004-09-14

    A portable, hand-held meter used to measure direct current (DC) attenuation in low impedance electrical signal cables and signal attenuators. A DC voltage is applied to the signal input of the cable and feedback to the control circuit through the signal cable and attenuators. The control circuit adjusts the applied voltage to the cable until the feedback voltage equals the reference voltage. The "units" of applied voltage required at the cable input is the system attenuation value of the cable and attenuators, which makes this meter unique. The meter may be used to calibrate data signal cables, attenuators, and cable-attenuator assemblies.

  10. Aerobic exercise training increases circulating insulin-like growth factor binding protein-1 concentration, but does not attenuate the reduction in circulating insulin-like growth factor binding protein-1 after a high-fat meal.

    PubMed

    Prior, Steven J; Jenkins, Nathan T; Brandauer, Josef; Weiss, Edward P; Hagberg, James M

    2012-03-01

    Insulin-like growth factor binding protein-1 (IGFBP-1) has metabolic effects throughout the body, and its expression is regulated in part by insulin. Circulating IGFBP-1 predicts development of cardiometabolic diseases in longitudinal studies, and low IGFBP-1 concentrations are associated with insulin resistance and consumption of a high-fat diet. Because of the favorable metabolic effects of regular aerobic exercise, we hypothesized that aerobic exercise training would increase plasma IGFBP-1 concentrations and attenuate the reduction in IGFBP-1 after a high-fat meal. Ten overweight (body mass index = 28.7 ± 0.9 kg/m(2)), older (61 ± 2 years) men and women underwent high-fat feeding and oral glucose tolerance tests at baseline and after 6 months of aerobic exercise training. In response to aerobic exercise training, subjects increased cardiorespiratory fitness by 13% (P < .05) and insulin sensitivity index by 28% (P < .05). Basal plasma concentrations of IGFBP-1 increased by 41% after aerobic exercise training (P < .05). The insulin response to an oral glucose tolerance test was a significant predictor of fasting plasma IGFBP-1 concentrations at baseline and after exercise training (P = .02). In response to the high-fat meal at baseline, plasma IGFBP-1 concentrations decreased by 58% (P < .001); a 61% decrease to similar postprandial concentrations was observed after exercise training (P < .001). Plasma insulin response to the high-fat meal was inversely associated with postprandial IGFBP-1 concentrations at baseline and after exercise training (P = .06 and P < .05, respectively). Although aerobic exercise training did not attenuate the response to a high-fat meal, the increase in IGFBP-1 concentrations after exercise training may be one mechanism by which exercise reduces risk for cardiometabolic diseases in older adults.

  11. The SopEPhi phage integrates into the ssrA gene of Salmonella enterica serovar Typhimurium A36 and is closely related to the Fels-2 prophage.

    PubMed

    Pelludat, Cosima; Mirold, Susanne; Hardt, Wolf-Dietrich

    2003-09-01

    Salmonella spp. are enteropathogenic gram-negative bacteria that use a large array of virulence factors to colonize the host, manipulate host cells, and resist the host's defense mechanisms. Even closely related Salmonella strains have different repertoires of virulence factors. Bacteriophages contribute substantially to this diversity. There is increasing evidence that the reassortment of virulence factor repertoires by converting phages like the GIFSY phages and SopEPhi may represent an important mechanism in the adaptation of Salmonella spp. to specific hosts and to the emergence of new epidemic strains. Here, we have analyzed in more detail SopEPhi, a P2-like phage from Salmonella enterica serovar Typhimurium DT204 that encodes the virulence factor SopE. We have cloned and characterized the attachment site (att) of SopEPhi and found that its 47-bp core sequence overlaps the 3' terminus of the ssrA gene of serovar Typhimurium. Furthermore, we have demonstrated integration of SopEPhi into the cloned attB site of serovar Typhimurium A36. Sequence analysis of the plasmid-borne prophage revealed that SopEPhi is closely related to (60 to 100% identity over 80% of the genome) but clearly distinct from the Fels-2 prophage of serovar Typhimurium LT2 and from P2-like phages in the serovar Typhi CT18 genome. Our results demonstrate that there is considerable variation among the P2-like phages present in closely related Salmonella spp.

  12. Plasmids and packaging cell lines for use in phage display

    DOEpatents

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  13. Complete Genome Sequence of the Streptomyces Phage Nanodon

    PubMed Central

    2016-01-01

    Streptomyces phage Nanodon is a temperate double-stranded DNA Siphoviridae belonging to cluster BD1. It was isolated from soil collected in Kilauea, HI, using Streptomyces griseus subsp. griseus as a host. PMID:27795236

  14. Sequence analysis of the Lactobacillus temperate phage Sha1.

    PubMed

    Yoon, Bo Hyun; Jang, Se Hwan; Chang, Hyo-Ihl

    2011-09-01

    Bacteriophage Sha1, a newly isolated temperate phage from a mitomycin-C-induced lysate of Lactobacillus plantarum isolated from Kimchi, has an isometric head (58 nm × 60 nm) and a long tail (259 nm × 11 nm). The double-strand DNA genome of the phage Sha1 was 41,726 base pairs (bp) long, with a G+C content of 40.61%. Bioinformatic analysis of Sha1 shows that this phage contains 58 putative open reading frames (ORFs). Sha1 can be classified as a member of the large family Siphoviridae by genomic structure and morphology. To our knowledge, this is the first report of genomic sequencing and characterization of temperate phage Sha1 from wild-type L. plantarum isolated from kimchi in Korea. PMID:21701917

  15. [Inactivation of T4 phage in water environment using proteinase].

    PubMed

    Lü, Wen-zhou; Yang, Qing-xiang; Zhang, Yu; Yang, Min; Zhu, Chun-fang

    2004-09-01

    The inactivation effectiveness of proteinase to viruses was investigated by using T4 phage as a model virus. The results showed that the inactivation effectiveness of proteinase to T4 phage was obvious. In the optimum conditions and 67.5 u/mL concentration, the inactivation rate of proteinase K to T4 phage in sterilized water and in sewage achieved 99.4% and 49.4% respectively in an hour, and achieved >99.9% and 81.1% in three hours. The inactivation rate of the industrial proteinase 1398 to T4 phage in sterilized water achieved 74.4% in an hour. The effects of pH and temperature on the inactivation effectiveness was not evident.

  16. Phage display creates innovative applications to combat hepatitis B virus

    PubMed Central

    Tan, Wen Siang; Ho, Kok Lian

    2014-01-01

    Hepatitis B virus (HBV) has killed countless lives in human history. The invention of HBV vaccines in the 20th century has reduced significantly the rate of the viral infection. However, currently there is no effective treatment for chronic HBV carriers. Newly emerging vaccine escape mutants and drug resistant strains have complicated the viral eradication program. The entire world is now facing a new threat of HBV and human immunodeficiency virus co-infection. Could phage display provide solutions to these life-threatening problems? This article reviews critically and comprehensively the innovative and potential applications of phage display in the development of vaccines, therapeutic agents, diagnostic reagents, as well as gene and drug delivery systems to combat HBV. The application of phage display in epitope mapping of HBV antigens is also discussed in detail. Although this review mainly focuses on HBV, the innovative applications of phage display could also be extended to other infectious diseases. PMID:25206271

  17. Salmonella phages and prophages--genomics and practical aspects.

    PubMed

    Kropinski, Andrew M; Sulakvelidze, Alexander; Konczy, Paulina; Poppe, Cornelius

    2007-01-01

    Numerous bacteriophages specific to Salmonella have been isolated or identified as part of host genome sequencing projects. Phylogenetic analysis of the sequenced phages, based on related protein content using CoreGenes, reveals that these viruses fall into five groupings (P27-like, P2-like, lambdoid, P22-like, and T7-like) and three outliers (epsilon15, KS7, and Felix O1). The P27 group is only represented by ST64B; the P2 group contains Fels-2, SopEphi, and PSP3; the lambdoid Salmonella phages include Gifsy-1, Gifsy-2, and Fels-1. The P22-like viruses include epsilon34, ES18, P22, ST104, and ST64T. The only member of the T7-like group is SP6. The properties of each of these phages are discussed, along with their role as agents of genetic exchange and as therapeutic agents and their involvement in phage typing.

  18. Attenuation of free spheroidal oscillations of the Earth after the M = 9 earthquake in Sumatra and super-deep earthquake in the Sea of Okhotsk: II. interpretation of the observed Q-factor

    NASA Astrophysics Data System (ADS)

    Molodenskii, S. M.; Molodenskii, M. S.

    2015-11-01

    In the first part of the paper, the range of the admissible values of the Q-factor for the fundamental spheroidal modes and overtones was calculated from the records of the free oscillations of the Earth after the earthquake with M = 9 in Sumatra and the super-deep earthquake in the Sea of Okhotsk. Below, the interpretation of the data obtained in the first part of the paper is presented. By orthogonalization of the functional derivatives of the eigenfrequencies with respect to the density and Q-factor of the mantle, the model distributions of these parameters which best fit the whole set of the data about the attenuation of the free oscillations and the phases of forced nutations of the Earth are reconstructed. The use of the attenuation data for the free oscillations recorded after the super-deep earthquake in the Sea of Okhotsk on May 24, 2013 significantly improves the accuracy of the Q-factor reconstruction at different depths in the mantle. The implications of the free oscillations' attenuation data for the solution of the inverse problem of reconstructing the profiles of density and creep function of the mantle in the interval of periods from 1 s to one day are studied. Without the allowance for the attenuation data, the reconstruction errors for the density profiles were about 0.1 g/cm3, and for the shear moduli at the oscillation period of 200 s, about 4 × 109 dyn/cm2. The use of the free oscillation attenuation data largely removes this uncertainty. Although the relative measurement accuracy of the Q-factor is by about two orders of magnitude lower than the measurement accuracy of all eigenfrequencies, the weights of relative residuals of Q in the minimand functional of the weighted mean square deviations should be of the same order of magnitude as the weights for the relative changes in the free oscillation frequencies. With the allowance for the new attenuation data obtained in the first part of the paper, the reconstruction errors for these parameters

  19. Deep sequencing analysis of phage libraries using Illumina platform.

    PubMed

    Matochko, Wadim L; Chu, Kiki; Jin, Bingjie; Lee, Sam W; Whitesides, George M; Derda, Ratmir

    2012-09-01

    This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 10(7) reads in a single run. We applied Illumina sequencing to analyze phage libraries. Using PCR, we isolated the variable regions from M13KE phage vectors from a phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5'-terminal region. We used this approach to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2×10(7) reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6bp), one complimentary region (12bp) and a variable region (36bp). We applied this sequencing to a model library of 10(6) unique clones and observed that amplification enriches ∼150 clones, which dominate ∼20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in phage screens. It could also be instrumental in developing new screening strategies, which can preserve

  20. Comparative genomics and stx phage characterization of LEE-negative Shiga toxin-producing Escherichia coli.

    PubMed

    Steyert, Susan R; Sahl, Jason W; Fraser, Claire M; Teel, Louise D; Scheutz, Flemming; Rasko, David A

    2012-01-01

    Infection by Escherichia coli and Shigella species are among the leading causes of death due to diarrheal disease in the world. Shiga toxin-producing E. coli (STEC) that do not encode the locus of enterocyte effacement (LEE-negative STEC) often possess Shiga toxin gene variants and have been isolated from humans and a variety of animal sources. In this study, we compare the genomes of nine LEE-negative STEC harboring various stx alleles with four complete reference LEE-positive STEC isolates. Compared to a representative collection of prototype E. coli and Shigella isolates representing each of the pathotypes, the whole genome phylogeny demonstrated that these isolates are diverse. Whole genome comparative analysis of the 13 genomes revealed that in addition to the absence of the LEE pathogenicity island, phage-encoded genes including non-LEE encoded effectors, were absent from all nine LEE-negative STEC genomes. Several plasmid-encoded virulence factors reportedly identified in LEE-negative STEC isolates were identified in only a subset of the nine LEE-negative isolates further confirming the diversity of this group. In combination with whole genome analysis, we characterized the lambdoid phages harboring the various stx alleles and determined their genomic insertion sites. Although the integrase gene sequence corresponded with genomic location, it was not correlated with stx variant, further highlighting the mosaic nature of these phages. The transcription of these phages in different genomic backgrounds was examined. Expression of the Shiga toxin genes, stx(1) and/or stx(2), as well as the Q genes, were examined with quantitative reverse transcriptase polymerase chain reaction assays. A wide range of basal and induced toxin induction was observed. Overall, this is a first significant foray into the genome space of this unexplored group of emerging and divergent pathogens.

  1. Imaging Rayleigh wave attenuation with USArray

    NASA Astrophysics Data System (ADS)

    Bao, Xueyang; Dalton, Colleen A.; Jin, Ge; Gaherty, James B.; Shen, Yang

    2016-07-01

    The EarthScope USArray provides an opportunity to obtain detailed images of the continental upper mantle at an unprecedented scale. The majority of mantle models derived from USArray data to date contain spatial variations in seismic-wave speed; however, in many cases these data sets do not by themselves allow a non-unique interpretation. Joint interpretation of seismic attenuation and velocity models can improve upon the interpretations based only on velocity and provide important constraints on the temperature, composition, melt content, and volatile content of the mantle. The surface wave amplitudes that constrain upper-mantle attenuation are sensitive to factors in addition to attenuation, including the earthquake source excitation, focusing and defocusing by elastic structure, and local site amplification. Because of the difficulty of isolating attenuation from these other factors, little is known about the attenuation structure of the North American upper mantle. In this study, Rayleigh wave traveltime and amplitude in the period range 25-100 s are measured using an interstation cross-correlation technique, which takes advantage of waveform similarity at nearby stations. Several estimates of Rayleigh wave attenuation and site amplification are generated at each period, using different approaches to separate the effects of attenuation and local site amplification on amplitude. It is assumed that focusing and defocusing effects can be described by the Laplacian of the traveltime field. All approaches identify the same large-scale patterns in attenuation, including areas where the attenuation values are likely contaminated by unmodelled focusing and defocusing effects. Regionally averaged attenuation maps are constructed after removal of the contaminated attenuation values, and the variations in intrinsic shear attenuation that are suggested by these Rayleigh wave attenuation maps are explored.

  2. Properties of Klebsiella phage P13 and associated exopolysaccharide depolymerase

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Li, Guiyang; Mo, Zhaolan; Chai, Zihan; Shang, Anqi; Mou, Haijin

    2013-11-01

    The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide (EPS) produced by this microorganism. In this study, the titer of phage P13, initially 2.0 × 107 pfu mL-1, was found increasing 20 min after infection and reached 5.0 × 109 pfu mL-1 in 60 min. Accordingly, the enzyme activity of depolymerase approached the maximum 60 min after infection. Treatment at 70°C for 30 min inactivated all the phage, but retained over 90% of the depolymerase activity. Addition of acetone into the crude phage lysate led to precipitation of the protein, with a marked increase in bacterial EPS degradation activity and a rapid drop in the titer of phage. After partial purification by acetone precipitation and ultrafiltration centrifugation, the enzyme was separated from the phage particles, showing two components with enzyme activity on Q-Sepharose Fast Flow. The soluble enzyme had an optimum degradation activity at 60°C and pH 6.5. Transmission electron microscopy demonstrated that the phage P13 particles were spherical with a diameter of 50 nm and a short stumpy tail. It was a doublestrand DNA virus consisting of a nucleic acid molecule of 45976 bp. This work provides an efficient purification operation including thermal treatment and ultrafiltration centrifugation, to dissociate depolymerase from phage particles. The characterization of phage P13 and associated EPS depolymerase is beneficial for further application of this enzyme.

  3. Molecular architecture of tailed double-stranded DNA phages

    PubMed Central

    Fokine, Andrei; Rossmann, Michael G

    2014-01-01

    The tailed double-stranded DNA bacteriophages, or Caudovirales, constitute ~96% of all the known phages. Although these phages come in a great variety of sizes and morphology, their virions are mainly constructed of similar molecular building blocks via similar assembly pathways. Here we review the structure of tailed double-stranded DNA bacteriophages at a molecular level, emphasizing the structural similarity and common evolutionary origin of proteins that constitute these virions. PMID:24616838

  4. Primary Isolation Strain Determines Both Phage Type and Receptors Recognised by Campylobacter jejuni Bacteriophages

    PubMed Central

    Sørensen, Martine C. Holst; Gencay, Yilmaz Emre; Birk, Tina; Baldvinsson, Signe Berg; Jäckel, Claudia; Hammerl, Jens A.; Vegge, Christina S.; Neve, Horst; Brøndsted, Lone

    2015-01-01

    In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS) for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN) of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb), host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220) as well as receptors (CPS or flagella) recognised by the isolated phages. PMID:25585385

  5. The Legacy of 20th Century Phage Research.

    PubMed

    Campbell, Allan M

    2010-09-01

    The Golden Age of Phage Research, where phage was the favored material for attacking many basic questions in molecular biology, lasted from about 1940 to 1970. The era was initiated by Ellis and Delbrück, whose analysis defined the relevant parameters to measure in studying phage growth, and depended on the fact that the contents of a plaque can comprise descendants of a single infecting particle. It ended around 1970 because definitive methods had then become available for answering the same questions in other systems. Some of the accomplishments of phage research were the demonstration by Hershey and Chase that the genetic material of phage T2 is largely composed of DNA, the construction of linkage maps of T2 and T4 by Hershey and Rotman and their extension to very short molecular distances by Benzer, and the isolation of conditionally lethal mutants in T4 by Epstein et al. and in λ by Campbell. The dissection of the phage life cycle into causal chains was explored by Edgar and Wood for T4 assembly and later in the regulation of lysogeny by Kaiser, extended to the molecular level by Ptashne and others. Restriction/modification was discovered in λ by Bertani and Weigle, and the biochemical mechanism was elucidated by Arber and by Smith.

  6. Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

    PubMed Central

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; Liang, Tiffany Y.; Pivaroff, Cullen G.; Haynes, Matthew R.; Nulton, Jim; Felts, Ben; Bailey, Barbara A.; Salamon, Peter; Edwards, Robert A.; Burgin, Alex B.; Segall, Anca M.; Rohwer, Forest

    2015-01-01

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented. PMID:26132888

  7. Phage therapy against Enterococcus faecalis in dental root canals.

    PubMed

    Khalifa, Leron; Shlezinger, Mor; Beyth, Shaul; Houri-Haddad, Yael; Coppenhagen-Glazer, Shunit; Beyth, Nurit; Hazan, Ronen

    2016-01-01

    Antibiotic resistance is an ever-growing problem faced by all major sectors of health care, including dentistry. Recurrent infections related to multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, and vancomycin-resistant enterococci (VRE) in hospitals are untreatable and question the effectiveness of notable drugs. Two major reasons for these recurrent infections are acquired antibiotic resistance genes and biofilm formation. None of the traditionally known effective techniques have been able to efficiently resolve these issues. Hence, development of a highly effective antibacterial practice has become inevitable. One example of a hard-to-eradicate pathogen in dentistry is Enterococcus faecalis, which is one of the most common threats observed in recurrent root canal treatment failures, of which the most problematic to treat are its biofilm-forming VRE strains. An effective response against such infections could be the use of bacteriophages (phages). Phage therapy was found to be highly effective against biofilm and multidrug-resistant bacteria and has other advantages like ease of isolation and possibilities for genetic manipulations. The potential of phage therapy in dentistry, in particular against E. faecalis biofilms in root canals, is almost unexplored. Here we review the efforts to develop phage therapy against biofilms. We also focus on the phages isolated against E. faecalis and discuss the possibility of using phages against E. faecalis biofilm in root canals. PMID:27640530

  8. Experimental phage therapy of burn wound infection: difficult first steps

    PubMed Central

    Rose, Thomas; Verbeken, Gilbert; Vos, Daniel De; Merabishvili, Maya; Vaneechoutte, Mario; Lavigne, Rob; Jennes, Serge; Zizi, Martin; Pirnay, Jean-Paul

    2014-01-01

    Antibiotic resistance has become a major public health problem and the antibiotics pipeline is running dry. Bacteriophages (phages) may offer an ‘innovative’ means of infection treatment, which can be combined or alternated with antibiotic therapy and may enhance our abilities to treat bacterial infections successfully. Today, in the Queen Astrid Military Hospital, phage therapy is increasingly considered as part of a salvage therapy for patients in therapeutic dead end, particularly those with multidrug resistant infections. We describe the application of a well-defined and quality controlled phage cocktail, active against Pseudomonas aeruginosa and Staphylococcus aureus, on colonized burn wounds within a modest clinical trial (nine patients, 10 applications), which was approved by a leading Belgian Medical Ethical Committee. No adverse events, clinical abnormalities or changes in laboratory test results that could be related to the application of phages were observed. Unfortunately, this very prudent ‘clinical trial’ did not allow for an adequate evaluation of the efficacy of the phage cocktail. Nevertheless, this first ‘baby step’ revealed several pitfalls and lessons for future experimental phage therapy and helped overcome the psychological hurdles that existed to the use of viruses in the treatment of patients in our burn unit. PMID:25356373

  9. Conserved termini and adjacent variable region of Twortlikevirus Staphylococcus phages.

    PubMed

    Zhang, Xianglilan; Kang, Huaixing; Li, Yuyuan; Liu, Xiaodong; Yang, Yu; Li, Shasha; Pei, Guangqian; Sun, Qiang; Shu, Peng; Mi, Zhiqiang; Huang, Yong; Zhang, Zhiyi; Liu, Yannan; An, Xiaoping; Xu, Xiaolu; Tong, Yigang

    2015-12-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing cause of serious infection, both in the community and hospital settings. Despite sophisticated strategies and efforts, the antibiotic options for treating MRSA infection are narrowing because of the limited number of newly developed antimicrobials. Here, four newly-isolated MRSA-virulent phages, IME-SA1, IMESA2, IME-SA118 and IME-SA119, were sequenced and analyzed. Their genome termini were identified using our previously proposed "termini analysis theory". We provide evidence that remarkable conserved terminus sequences are found in IME-SA1/2/118/119, and, moreover, are widespread throughout Twortlikevirus Staphylococcus phage G1 and K species. Results also suggested that each phage of the two species has conserved 5' terminus while the 3' terminus is variable. More importantly, a variable region with a specific pattern was found to be present near the conserved terminus of Twortlikevirus S. phage G1 species. The clone with the longest variable region had variable terminus lengths in successive generations, while the clones with the shortest variable region and with the average length variable region maintained the same terminal length as themselves during successive generations. IME-SA1 bacterial infection experiments showed that the variation is not derived from adaptation of the phage to different host strains. This is the first study of the conserved terminus and variable region of Twortlikevirus S. phages.

  10. Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa.

    PubMed

    Chan, Benjamin K; Sistrom, Mark; Wertz, John E; Kortright, Kaitlyn E; Narayan, Deepak; Turner, Paul E

    2016-01-01

    Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies. Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections. PMID:27225966

  11. Phage therapy against Enterococcus faecalis in dental root canals

    PubMed Central

    Khalifa, Leron; Shlezinger, Mor; Beyth, Shaul; Houri-Haddad, Yael; Coppenhagen-Glazer, Shunit; Beyth, Nurit; Hazan, Ronen

    2016-01-01

    Antibiotic resistance is an ever-growing problem faced by all major sectors of health care, including dentistry. Recurrent infections related to multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, and vancomycin-resistant enterococci (VRE) in hospitals are untreatable and question the effectiveness of notable drugs. Two major reasons for these recurrent infections are acquired antibiotic resistance genes and biofilm formation. None of the traditionally known effective techniques have been able to efficiently resolve these issues. Hence, development of a highly effective antibacterial practice has become inevitable. One example of a hard-to-eradicate pathogen in dentistry is Enterococcus faecalis, which is one of the most common threats observed in recurrent root canal treatment failures, of which the most problematic to treat are its biofilm-forming VRE strains. An effective response against such infections could be the use of bacteriophages (phages). Phage therapy was found to be highly effective against biofilm and multidrug-resistant bacteria and has other advantages like ease of isolation and possibilities for genetic manipulations. The potential of phage therapy in dentistry, in particular against E. faecalis biofilms in root canals, is almost unexplored. Here we review the efforts to develop phage therapy against biofilms. We also focus on the phages isolated against E. faecalis and discuss the possibility of using phages against E. faecalis biofilm in root canals. PMID:27640530

  12. Phage phenomics: Physiological approaches to characterize novel viral proteins

    DOE PAGES

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; Liang, Tiffany Y.; Pivaroff, Cullen G.; Haynes, Matthew R.; Nulton, Jim; Felts, Ben; Bailey, Barbara A.; Salamon, Peter; et al

    2015-06-11

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysismore » by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.« less

  13. Phage phenomics: Physiological approaches to characterize novel viral proteins

    SciTech Connect

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; Liang, Tiffany Y.; Pivaroff, Cullen G.; Haynes, Matthew R.; Nulton, Jim; Felts, Ben; Bailey, Barbara A.; Salamon, Peter; Edwards, Robert A.; Burgin, Alex B.; Segall, Anca M.; Rohwer, Forest

    2015-06-11

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

  14. Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa

    PubMed Central

    Chan, Benjamin K.; Sistrom, Mark; Wertz, John E.; Kortright, Kaitlyn E.; Narayan, Deepak; Turner, Paul E.

    2016-01-01

    Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies. Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections. PMID:27225966

  15. The Shiga toxin 2 production level in enterohemorrhagic Escherichia coli O157:H7 is correlated with the subtypes of toxin-encoding phage.

    PubMed

    Ogura, Yoshitoshi; Mondal, Shakhinur Islam; Islam, Md Rakibul; Mako, Toshihiro; Arisawa, Kokichi; Katsura, Keisuke; Ooka, Tadasuke; Gotoh, Yasuhiro; Murase, Kazunori; Ohnishi, Makoto; Hayashi, Tetsuya

    2015-11-16

    Enterohemorrhagic E. coli (EHEC) causes diarrhea and hemorrhagic colitis with life-threatening complications, such as hemolytic uremic syndrome. Their major virulence factor is Shiga toxin (Stx), which is encoded by bacteriophages. Of the two types of Stx, the production of Stx2, particularly that of Stx2a (a subtype of Stx2), is a major risk factor for severe EHEC infections, but the Stx2 production level is highly variable between strains. Here, we define four major and two minor subtypes of Stx2a-encoding phages according to their replication proteins. The subtypes are correlated with Stx2a titers produced by the host O157 strains, suggesting a critical role of the phage subtype in determining the Stx2a production level. We further show that one of the two subclades in the clade 8, a proposed hyper-virulent lineage of O157, carries the Stx2 phage subtype that confers the highest Stx2 production to the host strain. The presence of this subclade may explain the proposed high virulence potential of clade 8. These results provide novel insights into the variation in virulence among O157 strains and highlight the role of phage variation in determining the production level of the virulence factors that phages encode.

  16. Evolutionary consequences of intra-patient phage predation on microbial populations.

    PubMed

    Seed, Kimberley D; Yen, Minmin; Shapiro, B Jesse; Hilaire, Isabelle J; Charles, Richelle C; Teng, Jessica E; Ivers, Louise C; Boncy, Jacques; Harris, Jason B; Camilli, Andrew

    2014-08-26

    The impact of phage predation on bacterial pathogens in the context of human disease is not currently appreciated. Here, we show that predatory interactions of a phage with an important environmentally transmitted pathogen, Vibrio cholerae, can modulate the evolutionary trajectory of this pathogen during the natural course of infection within individual patients. We analyzed geographically and temporally disparate cholera patient stool samples from Haiti and Bangladesh and found that phage predation can drive the genomic diversity of intra-patient V. cholerae populations. Intra-patient phage-sensitive and phage-resistant isolates were isogenic except for mutations conferring phage resistance, and moreover, phage-resistant V. cholerae populations were composed of a heterogeneous mix of many unique mutants. We also observed that phage predation can significantly alter the virulence potential of V. cholerae shed from cholera patients. We provide the first molecular evidence for predatory phage shaping microbial community structure during the natural course of infection in humans.

  17. Digitally Controlled Beam Attenuator

    NASA Astrophysics Data System (ADS)

    Peppler, W. W.; Kudva, B.; Dobbins, J. T.; Lee, C. S.; Van Lysel, M. S.; Hasegawa, B. H.; Mistretta, C. A.

    1982-12-01

    In digital fluorographic techniques the video camera must accommodate a wide dynamic range due to the large variation in the subject thickness within the field of view. Typically exposure factors and the optical aperture are selected such that the maximum video signal is obtained in the most transmissive region of the subject. Consequently, it has been shown that the signal-to-noise ratio is severely reduced in the dark regions. We have developed a prototype digital beam attenuator (DBA) which will alleviate this and some related problems in digital fluorography. The prototype DBA consists of a 6x6 array of pistons which are individually controlled. A membrane containing an attenuating solu-tion of (CeC13) in water and the piston matrix are placed between the x-ray tube and the subject. Under digital control the pistons are moved into the attenuating material in order to adjust the beam intensity over each of the 36 cells. The DBA control unit which digitizes the image during patient positioning will direct the pistons under hydraulic control to produce a uniform x-ray field exiting the subject. The pistons were designed to produce very little structural background in the image. In subtraction studies any structure would be cancelled. For non-subtraction studies such as cine-cardiology we are considering higher cell densities (eg. 64x64). Due to the narrow range of transmission provided by the DBA, in such studies ultra-high contrast films could be used to produce a high resolution quasi-subtraction display. Additional benefits of the DBA are: 1) reduced dose to the bright image areas when the dark areas are properly exposed. 2) improved scatter and glare to primary ratios, leading to improved contrast in the dark areas.

  18. Nanoscale bacteriophage biosensors beyond phage display.

    PubMed

    Lee, Jong-Wook; Song, Jangwon; Hwang, Mintai P; Lee, Kwan Hyi

    2013-01-01

    Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology.

  19. Nanoscale bacteriophage biosensors beyond phage display

    PubMed Central

    Lee, Jong-Wook; Song, Jangwon; Hwang, Mintai P; Lee, Kwan Hyi

    2013-01-01

    Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology. PMID:24143096

  20. Pressure surge attenuator

    DOEpatents

    Christie, Alan M.; Snyder, Kurt I.

    1985-01-01

    A pressure surge attenuation system for pipes having a fluted region opposite crushable metal foam. As adapted for nuclear reactor vessels and heads, crushable metal foam is disposed to attenuate pressure surges.

  1. The ltp gene of temperate Streptococcus thermophilus phage TP-J34 confers superinfection exclusion to Streptococcus thermophilus and Lactococcus lactis

    SciTech Connect

    Sun Xingmin . E-mail: Xingmin_Sun@brown.edu; Goehler, Andre; Heller, Knut J. . E-mail: knut.heller@bfel.de; Neve, Horst

    2006-06-20

    The ltp gene, located within the lysogeny module of temperate Streptococcus thermophilus phage TP-J34, has been shown to be expressed in lysogenic strain S. thermophilus J34. It codes for a lipoprotein, as demonstrated by inhibition of cleavage of the signal sequence by globomycin. Exposure of Ltp on the surface of Lactococcus lactis protoplasts bearing a plasmid-encoded copy of ltp has been demonstrated by immunogold labeling and electron microscopy. Expression of ltp in prophage- and plasmid-cured S. thermophilus J34-6f interfered with TP-J34 infection. While plating efficiency was reduced by a factor of about 40 and lysis of strain J34-6f in liquid medium was delayed considerably, phage adsorption was not affected at all. Intracellular accumulation of phage DNA was shown to be inhibited by Ltp. This indicates interference of Ltp with infection at the stage of triggering DNA release and injection into the cell, indicating a role of Ltp in superinfection exclusion. Expression of ltp in L. lactis Bu2-60 showed that the same superinfection exclusion mechanism was strongly effective against phage P008, a member of the lactococcal 936 phage species: no plaque-formation was detectable with even 10{sup 9} phage per ml applied, and lysis in liquid medium did not occur. In Lactococcus also, Ltp apparently inhibited phage DNA release and/or injection. Ltp appears to be a member of a family of small, secreted proteins with a 42 amino acids repeat structure encoded by genes of Gram-positive bacteria. Some of these homologous genes are part of the genomes of prophages.

  2. Bacteriophage exploitation of bacterial biofilms: phage preference for less mature targets?

    PubMed

    Abedon, Stephen T

    2016-02-01

    Robust evidence is somewhat lacking for biofilm susceptibility to bacteriophages in nature, contrasting often substantial laboratory biofilm vulnerability to phages. To help bridge this divide, I review a two-part scenario for 'heterogeneous' phage interaction even with phage-permissive single-species biofilms. First, through various mechanisms, those bacteria which are both more newly formed and located at biofilm surfaces may be particularly vulnerable to phage adsorption, rather than biofilm matrix being homogeneously resistant to phage penetration. Second, though phage infection of older, less metabolically active bacteria may still be virion productive, nevertheless the majority of phage population growth in association with biofilm bacteria could involve infection particularly of those bacteria which are more metabolically active and thereby better able to support larger phage bursts, versus clonally related biofilm bacteria equivalently supporting phage production. To the extent that biofilms are physiologically or structurally heterogeneous, with phages exploiting particularly relatively newly divided biofilm-surface bacteria, then even effective phage predation of natural biofilms could result in less than complete overall biofilm clearance. Phage tendencies toward only partial exploitation of even single-species biofilms could be consistent with observations that chronic bacterial infections in the clinic can require more aggressive or extensive phage therapy to eradicate.

  3. Proteomic Analysis of a Novel Bacillus Jumbo Phage Revealing Glycoside Hydrolase As Structural Component.

    PubMed

    Yuan, Yihui; Gao, Meiying

    2016-01-01

    Tailed phages with genomes of larger than 200 kbp are classified as Jumbo phages and exhibited extremely high uncharted diversity. The genomic annotation of Jumbo phage is often disappointing because most of the predicted proteins, including structural proteins, failed to make good hits to the sequences in the databases. In this study, 23 proteins of a novel Bacillus Jumbo phage, vB_BpuM_BpSp, were identified as phage structural proteins by the structural proteome analysis, including 14 proteins of unknown function, 5 proteins with predicted function as structural proteins, a glycoside hydrolase, a Holliday junction resolvase, a RNA-polymerase β-subunit, and a host-coding portal protein, which might be hijacked from the host strain during phage virion assembly. The glycoside hydrolase (Gp255) was identified as phage virion component and was found to interact with the phage baseplate protein. Gp255 shows specific lytic activity against the phage host strain GR8 and has high temperature tolerance. In situ peptidoglycan-hydrolyzing activities analysis revealed that the expressed Gp255 and phage structural proteome exhibited glycoside hydrolysis activity against the tested GR8 cell extracts. This study identified the first functional individual structural glycoside hydrolase in phage virion. The presence of activated glycoside hydrolase in phage virions might facilitate the injection of the phage genome during infection by forming pores on the bacterial cell wall. PMID:27242758

  4. Temperate phages both mediate and drive adaptive evolution in pathogen biofilms

    PubMed Central

    Davies, Emily V.; James, Chloe E.; Williams, David; O’Brien, Siobhan; Fothergill, Joanne L.; Haldenby, Sam; Paterson, Steve; Winstanley, Craig

    2016-01-01

    Temperate phages drive genomic diversification in bacterial pathogens. Phage-derived sequences are more common in pathogenic than nonpathogenic taxa and are associated with changes in pathogen virulence. High abundance and mobilization of temperate phages within hosts suggests that temperate phages could promote within-host evolution of bacterial pathogens. However, their role in pathogen evolution has not been experimentally tested. We experimentally evolved replicate populations of Pseudomonas aeruginosa with or without a community of three temperate phages active in cystic fibrosis (CF) lung infections, including the transposable phage, ɸ4, which is closely related to phage D3112. Populations grew as free-floating biofilms in artificial sputum medium, mimicking sputum of CF lungs where P. aeruginosa is an important pathogen and undergoes evolutionary adaptation and diversification during chronic infection. Although bacterial populations adapted to the biofilm environment in both treatments, population genomic analysis revealed that phages altered both the trajectory and mode of evolution. Populations evolving with phages exhibited a greater degree of parallel evolution and faster selective sweeps than populations without phages. Phage ɸ4 integrated randomly into the bacterial chromosome, but integrations into motility-associated genes and regulators of quorum sensing systems essential for virulence were selected in parallel, strongly suggesting that these insertional inactivation mutations were adaptive. Temperate phages, and in particular transposable phages, are therefore likely to facilitate adaptive evolution of bacterial pathogens within hosts. PMID:27382184

  5. Bacteriophage exploitation of bacterial biofilms: phage preference for less mature targets?

    PubMed

    Abedon, Stephen T

    2016-02-01

    Robust evidence is somewhat lacking for biofilm susceptibility to bacteriophages in nature, contrasting often substantial laboratory biofilm vulnerability to phages. To help bridge this divide, I review a two-part scenario for 'heterogeneous' phage interaction even with phage-permissive single-species biofilms. First, through various mechanisms, those bacteria which are both more newly formed and located at biofilm surfaces may be particularly vulnerable to phage adsorption, rather than biofilm matrix being homogeneously resistant to phage penetration. Second, though phage infection of older, less metabolically active bacteria may still be virion productive, nevertheless the majority of phage population growth in association with biofilm bacteria could involve infection particularly of those bacteria which are more metabolically active and thereby better able to support larger phage bursts, versus clonally related biofilm bacteria equivalently supporting phage production. To the extent that biofilms are physiologically or structurally heterogeneous, with phages exploiting particularly relatively newly divided biofilm-surface bacteria, then even effective phage predation of natural biofilms could result in less than complete overall biofilm clearance. Phage tendencies toward only partial exploitation of even single-species biofilms could be consistent with observations that chronic bacterial infections in the clinic can require more aggressive or extensive phage therapy to eradicate. PMID:26738755

  6. Wogonin prevents lipopolysaccharide-induced acute lung injury and inflammation in mice via peroxisome proliferator-activated receptor gamma-mediated attenuation of the nuclear factor-kappaB pathway.

    PubMed

    Yao, Jing; Pan, Di; Zhao, Yue; Zhao, Li; Sun, Jie; Wang, Yu; You, Qi-Dong; Xi, Tao; Guo, Qing-Long; Lu, Na

    2014-10-01

    Acute lung injury (ALI) from a variety of clinical disorders, characterized by diffuse inflammation, is a cause of acute respiratory failure that develops in patients of all ages. Previous studies reported that wogonin, a flavonoid-like chemical compound which was found in Scutellaria baicalensis, has anti-inflammatory effects in several inflammation models, but not in ALI. Here, the in vivo protective effect of wogonin in the amelioration of lipopolysaccharide (LPS) -induced lung injury and inflammation was assessed. In addition, the in vitro effects and mechanisms of wogonin were studied in the mouse macrophage cell lines Ana-1 and RAW264.7. In vivo results indicated that wogonin attenuated LPS-induced histological alterations. Peripheral blood leucocytes decreased in the LPS-induced group, which was ameliorated by wogonin. In addition, wogonin inhibited the production of several inflammatory cytokines, including tumour necrosis factor-α, interleukin-1β (IL-1β) and IL-6, in the bronchoalveolar lavage fluid and lung tissues after LPS challenge, while the peroxisome proliferator-activated receptor γ (PPARγ) inhibitor GW9662 reversed these effects. In vitro results indicated that wogonin significantly decreased the secretion of IL-6, IL-1β and tumour necrosis factor-α in Ana-1 and RAW264.7 cells, which was suppressed by transfection of PPARγ small interfering RNA and GW9662 treatment. Moreover, wogonin activated PPARγ, induced PPARγ-mediated attenuation of the nuclear translocation and the DNA-binding activity of nuclear factor-κB in vivo and in vitro. In conclusion, all of these results showed that wogonin may serve as a promising agent for the attenuation of ALI-associated inflammation and pathology by regulating the PPARγ-involved nuclear factor-κB pathway. PMID:24766487

  7. Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption

    PubMed Central

    Denes, Thomas; den Bakker, Henk C.; Tokman, Jeffrey I.; Guldimann, Claudia

    2015-01-01

    Listeria-infecting phages are readily isolated from Listeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface of Listeria monocytogenes strain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n = 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds to N-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminal N-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possess N-acetylglucosamine in their WTA. PMID:25888172

  8. Isolation and development of bioluminescent reporter phages for bacterial dysentery.

    PubMed

    Schofield, D A; Wray, D J; Molineux, I J

    2015-02-01

    Shigellosis is a significant cause of morbidity and mortality worldwide, most notably amongst children. Moreover, there is a global increase in the occurrence of multidrug-resistant isolates, including the epidemic and pandemic Shigella dysenteriae type 1 strain. We developed a bioluminescent reporter phage assay to facilitate detection and simultaneously determine antibiotic susceptibility. A Shigella flexneri phage (Shfl25875) was isolated from environmental wastewater and characterized by DNA sequencing. Shfl25875 is T4-like, harbors a 169,062-bp genome, and grows on most (28/29) S. flexneri strains and all 12 S. dysenteriae type 1 strains tested. The genes encoding bacterial luciferase were integrated into the Shfl25875 genome to create a "light-tagged" phage capable of transducing a bioluminescent phenotype to infected cells. Shfl25875::luxAB rapidly detects cultured isolates with high sensitivity. Specificity experiments indicate that the reporter does not respond to Shigella boydii, non-type 1 S. dysenteriae strains, and most non-Shigella Enterobacteriaceae. Shfl25875::luxAB generates ampicillin and ciprofloxacin susceptibility profiles that are similar to the standard Clinical and Laboratory Standards Institute (CLSI) growth microdilution method, but in a significantly shorter time. In addition, the reporter phage detects Shigella in mock-infected stool. This new reporter phage shows promise as a tool for the detection of cultured isolates or complex clinical samples.

  9. Topoisomerase Involvement in Multiplicity Reactivation of Phage T4

    PubMed Central

    Miskimins, Robin; Schneider, Silke; Johns, Virginia; Bernstein, Harris

    1982-01-01

    The products of phage T4 genes 39, 52 and probably 60 have been previously characterized as forming a type II DNA topoisomerase. Other evidence suggested that this topoisomerase promotes normal initiation of DNA replication, and that when it is defective its loss is partially compensated for by the host gyrase. We present evidence here that mutants defective in genes 39, 52 and 60 have reduced ability to carry out multiplicity reactivation (MR, a form of recombinational repair) of phage DNA damaged either by mitomycin C (MMC) or psoralen plus near-UV light (PUVA). We also observed that there is not extensive superhelicity in the intracellular phage DNA either in the presence or absence of the phage topoisomerase. This tends to rule out the possibility that the topoisomerase influences MR by controlling the general superhelicity of the phage DNA. The dependence of MR on topoisomerase could occur in several possible ways. However, we favor the explanation that the lesions are bypassed by a postreplication recombinational repair process that is influenced by the topoisomerase through its role in initiating replication. PMID:6293912

  10. Discovery of internalizing antibodies to tumor antigens from phage libraries

    PubMed Central

    Zhou, Yu; Marks, James D

    2014-01-01

    Phage antibody technology can be used to generate human antibodies to essentially any antigen. Many therapeutic target antigens are cell surface receptors, which can be challenging targets for antibody generation. In addition, for many therapeutic applications, one needs antibodies that not only bind the cell surface receptor but that also are internalized into the cell upon binding. This allows use of the antibody to deliver a range of payloads into the cell to achieve a therapeutic effect. In this chapter we describe how human phage antibody libraries can be selected directly on tumor cell lines to generate antibodies that bind cell surface receptors and which upon binding are rapidly internalized into the cell. Specific protocols show how to: 1) directly select cell binding and internalizing antibodies from human phage antibody libraries; 2) screen the phage antibodies in a high throughput flow cytometry assay for binding to the tumor cell line used for selection; 3) identify the antigen bound by the phage antibody using immunoprecipitation and mass spectrometry; and 4) direct cell binding and internalizing selections to a specific tumor antigen by sequential selection on a tumor cell line followed by selection on yeast displaying the target tumor antigen on the yeast surface. PMID:22208981

  11. Tracer attenuation in groundwater

    NASA Astrophysics Data System (ADS)

    Cvetkovic, Vladimir

    2011-12-01

    The self-purifying capacity of aquifers strongly depends on the attenuation of waterborne contaminants, i.e., irreversible loss of contaminant mass on a given scale as a result of coupled transport and transformation processes. A general formulation of tracer attenuation in groundwater is presented. Basic sensitivities of attenuation to macrodispersion and retention are illustrated for a few typical retention mechanisms. Tracer recovery is suggested as an experimental proxy for attenuation. Unique experimental data of tracer recovery in crystalline rock compare favorably with the theoretical model that is based on diffusion-controlled retention. Non-Fickian hydrodynamic transport has potentially a large impact on field-scale attenuation of dissolved contaminants.

  12. Chromium(III) Binding Phage Screening for the Selective Adsorption of Cr(III) and Chromium Speciation.

    PubMed

    Yang, Ting; Zhang, Xiao-Yu; Zhang, Xiao-Xiao; Chen, Ming-Li; Wang, Jian-Hua

    2015-09-30

    The screening of suitable sorption medium is the key for highly selective solid phase extraction (SPE) of heavy metals. Herein, we demonstrate a universal protocol for producing selective SPE adsorbent through an evolutional approach based on phage display peptide library. By choosing chromium(III) as the model target, immobilized Cr(III) resins are first prepared using Ni-NTA affinity resins for the interaction with NEB heptapeptide phage library. After three rounds of positive biopanning against target Cr(III) and negative biopanning against foreign metal species, Cr(III) binding phages with high selectivity are obtained. The binding affinity and selectivity are further assessed with ELISA. The phages bearing peptide (YKASLIT) is finally chosen and immobilized on cytopore beads for Cr(III) preconcentration. The retained Cr(III) is efficiently recovered by 0.10 mol L(-1) HNO3 and quantified with ICP-MS. By loading 4000 μL of sample solution at pH 7.0 for 2 h and stripping with 400 μL of 0.10 mol L(-1) HNO3, a linear range of 0.05-0.50 μg L(-1) is achieved along with an enrichment factor of 7.1. The limit of detection is derived to be 15 ng L(-1) (3σ, n = 7) with a RSD of 3.6% (0.25 μg L(-1), n = 7). The procedure is validated by analyzing chromium content in a certified reference material GBW08608 (simulate water). In addition, chromium speciation in real water samples is demonstrated. Cr(VI) is first converted into Cr(III), and the latter subjected to the sorption onto the Cr(III) binding phage, followed by elution and quantification of the total chromium amount, and finally speciation is achieved by difference.

  13. Chromium(III) Binding Phage Screening for the Selective Adsorption of Cr(III) and Chromium Speciation.

    PubMed

    Yang, Ting; Zhang, Xiao-Yu; Zhang, Xiao-Xiao; Chen, Ming-Li; Wang, Jian-Hua

    2015-09-30

    The screening of suitable sorption medium is the key for highly selective solid phase extraction (SPE) of heavy metals. Herein, we demonstrate a universal protocol for producing selective SPE adsorbent through an evolutional approach based on phage display peptide library. By choosing chromium(III) as the model target, immobilized Cr(III) resins are first prepared using Ni-NTA affinity resins for the interaction with NEB heptapeptide phage library. After three rounds of positive biopanning against target Cr(III) and negative biopanning against foreign metal species, Cr(III) binding phages with high selectivity are obtained. The binding affinity and selectivity are further assessed with ELISA. The phages bearing peptide (YKASLIT) is finally chosen and immobilized on cytopore beads for Cr(III) preconcentration. The retained Cr(III) is efficiently recovered by 0.10 mol L(-1) HNO3 and quantified with ICP-MS. By loading 4000 μL of sample solution at pH 7.0 for 2 h and stripping with 400 μL of 0.10 mol L(-1) HNO3, a linear range of 0.05-0.50 μg L(-1) is achieved along with an enrichment factor of 7.1. The limit of detection is derived to be 15 ng L(-1) (3σ, n = 7) with a RSD of 3.6% (0.25 μg L(-1), n = 7). The procedure is validated by analyzing chromium content in a certified reference material GBW08608 (simulate water). In addition, chromium speciation in real water samples is demonstrated. Cr(VI) is first converted into Cr(III), and the latter subjected to the sorption onto the Cr(III) binding phage, followed by elution and quantification of the total chromium amount, and finally speciation is achieved by difference. PMID:26346061

  14. Nerve Growth Factor Protects the Ischemic Heart via Attenuation of the Endoplasmic Reticulum Stress Induced Apoptosis by Activation of Phosphatidylinositol 3-Kinase

    PubMed Central

    Wei, Ke; Liu, Li; Xie, Fei; Hao, Xuechao; Luo, Jie; Min, Su

    2015-01-01

    Background: Increased expression of nerve growth factor (NGF) has been found in the myocardium suffered from ischemia and reperfusion (I/R). The pro-survival activity of NGF on ischemic heart has been supposed to be mediated by phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Endoplasmic reticulum (ER) stress, which is activated initially as a defensive response to eliminate the accumulated unfolded proteins, has shown a critical involvement in the ischemia induced myocardial apoptosis. This study was aimed to investigate whether NGF induced heart protection against I/R injury includes a mechanism of attenuation of ER stress-induced myocardial apoptosis by activation of PI3K/Akt pathway. Methods: Isolated adult rat hearts were perfused with a Langendörff perfusion system. Hearts in the Sham group were subjected to 225 min of continuous Krebs-Henseleit buffer (KHB) perfusion without ischemia. Hearts in I/R group were perfused with KHB for a 75-min of equilibration period followed by 30 min of global ischemia and 120 min of KHB reperfusion. Hearts in the NGF group accepted 45 min of euilibration perfusion and 30 min of NGF pretreatment (with a final concentration of 100 ng/ml in the KHB) before 30 min of global ischemia and 120 min of reperfusion. Hearts in K252a and LY294002 groups were pretreated with either a TrkA inhibitor, K252a or a phosphatidyl inositol 3-kinase inhibitor, LY294002 for 30 min before NGF (100 ng/ml) administration. Cardiac hemodynamics were measured from the beginning of the perfusion. Cardiac enzymes and cardiac troponin I (cTnI) were assayed before ischemia and at the end of reperfusion. Myocardial apoptosis rate was measured by TUNEL staining, and expression of glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, total- and phospho-(Ser473)-Akt were assessed by Western blot analyses. Results: NGF pretreatment significantly improved the recovery of post

  15. Global Attenuation Model of the Upper Mantle

    NASA Astrophysics Data System (ADS)

    Adenis, A.; Debayle, E.; Ricard, Y. R.

    2015-12-01

    We present a three-dimensional shear attenuation model based on a massive surface wave data-set (372,629 Rayleigh waveforms analysed in the period range 50-300s by Debayle and Ricard, 2012). For each seismogram, this approach yields depth-dependent path average models of shear velocity and quality factor, and a set of fundamental and higher-mode dispersion and attenuation curves. We combine these attenuation measurements in a tomographic inversion after a careful rejection of the noisy data. We first remove data likely to be biased by a poor knowledge of the source. Then we assume that waves corresponding to events having close epicenters and recorded at the same station sample the same elastic and anelastic structure, we cluster the corresponding rays and average the attenuation measurements. Logarithms of the attenuations are regionalized using the non-linear east square formalism of Tarantola and Valette (1982), resulting in attenuation tomographic maps between 50s and 300s. After a first inversion, outlyers are rejected and a second inversion yields a moderate variance reduction of about 20%. We correct the attenuation curves for focusing effect using the linearized ray theory of Woodhouse and Wong (1986). Accounting for focussing effects allows building tomographic maps with variance reductions reaching 40%. In the period range 120-200s, the root mean square of the model perturbations increases from about 5% to 20%. Our 3-D attenuation models present strong agreement with surface tectonics at period lower than 200s. Areas of low attenuation are located under continents and areas of high attenuation are associated with oceans. Surprisingly, although mid oceanic ridges are located in attenuating regions, their signature, even if enhanced by focusing corrections, remains weaker than in the shear velocity models. Synthetic tests suggests that regularisation contributes to damp the attenuation signature of ridges, which could therefore be underestimated.

  16. Recent findings about the Yersinia enterocolitica phage shock protein response.

    PubMed

    Yamaguchi, Saori; Darwin, Andrew J

    2012-02-01

    The phage shock protein (Psp) system is a conserved extracytoplasmic stress response in bacteria that is essential for virulence of the human pathogen Yersinia enterocolitica. This article summarizes some recent findings about Y. enterocolitica Psp system function. Increased psp gene expression requires the transcription factor PspF, but under non-inducing conditions PspF is inhibited by an interaction with another protein, PspA, in the cytoplasm. A Psp-inducing stimulus causes PspA to relocate to the cytoplasmic membrane, freeing PspF to induce psp gene expression. This PspA relocation requires the integral cytoplasmic membrane proteins, PspB and PspC, which might sense an inducing trigger and sequester PspA by direct interaction. The subsequent induction of psp gene expression increases the PspA concentration, which also allows it to contact the membrane directly, perhaps for its physiological function. Mutational analysis of the PspB and PspC proteins has revealed that they both positively and negatively regulate psp gene expression and has also identified PspC domains associated with each function. We also compare the contrasting physiological roles of the Psp system in the virulence of Y. enterocolitica and Salmonella enterica sv. Typhimurium (S. Typhimurium). In S. Typhimurium, PspA maintains the proton motive force, which provides the energy needed to drive ion importers required for survival within macrophages. In contrast, in the extracellular pathogen Y. enterocolitica, PspB and PspC, but not PspA, are the Psp components needed for virulence. PspBC protect Y. enterocolitica from damage caused by the secretin component of its type 3 secretion system, an essential virulence factor.

  17. Measurement of the x-ray mass attenuation coefficient and determination of the imaginary component of the atomic form-factor of tin over the energy range of 29 keV-60 keV.

    SciTech Connect

    de Jonge, M. D.; Tran, C. Q.; Chantler, C. T.; Barnea, Z.; Dhal, B. P.; Paterson, D.; Kanter, E. P.; Southworth, S. H.; Young, L.; Beno, M. A.; Linton, J. A.; Jennings, G.; Univ. of Melbourne; Australian Synchrotron Project

    2007-01-01

    We use the x-ray extended-range technique (XERT) [C. T. Chantler et al., Phys. Rev. A 64, 062506 (2001)] to measure the mass attenuation coefficients of tin in the x-ray energy range of 29-60 keV to 0.04-3 % accuracy, and typically in the range 0.1-0.2 %. Measurements made over an extended range of the measurement parameter space are critically examined to identify, quantify, and correct a number of potential experimental systematic errors. These results represent the most extensive experimental data set for tin and include absolute mass attenuation coefficients in the regions of x-ray absorption fine structure, extended x-ray absorption fine structure, and x-ray absorption near-edge structure. The imaginary component of the atomic form factor f{sub 2} is derived from the photoelectric absorption after subtracting calculated Rayleigh and Compton scattering cross sections from the total attenuation. Comparison of the result with tabulations of calculated photoelectric absorption coefficients indicates that differences of 1-2 % persist between calculated and observed values.

  18. Abortive infection mechanisms and prophage sequences significantly influence the genetic makeup of emerging lytic lactococcal phages.

    PubMed

    Labrie, Simon J; Moineau, Sylvain

    2007-02-01

    In this study, we demonstrated the remarkable genome plasticity of lytic lactococcal phages that allows them to rapidly adapt to the dynamic dairy environment. The lytic double-stranded DNA phage ul36 was used to sequentially infect a wild-type strain of Lactococcus lactis and two isogenic derivatives with genes encoding two phage resistance mechanisms, AbiK and AbiT. Four phage mutants resistant to one or both Abi mechanisms were isolated. Comparative analysis of their complete genomes, as well as morphological observations, revealed that phage ul36 extensively evolved by large-scale homologous and nonhomologous recombination events with the inducible prophage present in the host strain. One phage mutant exchanged as much as 79% of its genome compared to the core genome of ul36. Thus, natural phage defense mechanisms and prophage elements found in bacterial chromosomes contribute significantly to the evolution of the lytic phage population.

  19. Understanding the enormous diversity of bacteriophages: the tailed phages that infect the bacterial family Enterobacteriaceae

    PubMed Central

    Grose, Julianne H.; Casjens, Sherwood R.

    2014-01-01

    Bacteriophages are the predominant biological entity on the planet. The recent explosion of sequence information has made estimates of their diversity possible. We describe the genomic comparison of 337 fully sequenced tailed phages isolated on 18 genera and 31 species of bacteria in the Enterobacteriaceae. These phages were largely unambiguously grouped into 56 diverse clusters (32 lytic and 24 temperate) that have syntenic similarity over >50% of the genomes within each cluster, but substantially less sequence similarity between clusters. Most clusters naturally break into sets of more closely related subclusters, 78% of which are correlated with their host genera. The largest groups of related phages are superclusters united by genome synteny to lambda (81 phages) and T7 (51 phages). This study forms a robust framework for understanding diversity and evolutionary relationships of existing tailed phages, for relating newly discovered phages and for determining host/phage relationships. PMID:25240328

  20. Evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage.

    PubMed

    Hendrix, R W; Smith, M C; Burns, R N; Ford, M E; Hatfull, G F

    1999-03-01

    We report DNA and predicted protein sequence similarities, implying homology, among genes of double-stranded DNA (dsDNA) bacteriophages and prophages spanning a broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections, not always direct, among the lambdoid phages of Escherichia coli, phage phiC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, phiflu, in the Haemophilus influenzae genome, and two small prophage-like elements, phiRv1 and phiRv2, in the genome of Mycobacterium tuberculosis. The results imply that these phage genes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA phage genomes are mosaics with access, by horizontal exchange, to a large common genetic pool but in which access to the gene pool is not uniform for all phage.

  1. Understanding the enormous diversity of bacteriophages: the tailed phages that infect the bacterial family Enterobacteriaceae.

    PubMed

    Grose, Julianne H; Casjens, Sherwood R

    2014-11-01

    Bacteriophages are the predominant biological entity on the planet. The recent explosion of sequence information has made estimates of their diversity possible. We describe the genomic comparison of 337 fully sequenced tailed phages isolated on 18 genera and 31 species of bacteria in the Enterobacteriaceae. These phages were largely unambiguously grouped into 56 diverse clusters (32 lytic and 24 temperate) that have syntenic similarity over >50% of the genomes within each cluster, but substantially less sequence similarity between clusters. Most clusters naturally break into sets of more closely related subclusters, 78% of which are correlated with their host genera. The largest groups of related phages are superclusters united by genome synteny to lambda (81 phages) and T7 (51 phages). This study forms a robust framework for understanding diversity and evolutionary relationships of existing tailed phages, for relating newly discovered phages and for determining host/phage relationships.

  2. A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display

    PubMed Central

    Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2013-01-01

    Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In ‘competitive phage display’ bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins. PMID:24225840

  3. A comparative study and phage typing of silage-making Lactobacillus bacteriophages.

    PubMed

    Doi, Katsumi; Zhang, Ye; Nishizaki, Yousuke; Umeda, Akiko; Ohmomo, Sadahiro; Ogata, Seiya

    2003-01-01

    To investigate basic characteristics of 10 virulent phages active on silage-making lactobacilli, morphological properties, host ranges, protein composition and genome characterization were separated into five groups based on host ranges and basic properties. The seven phages of groups I, II and V were active on Lactobacillus plantarum and Lactobacillus pentosus. Phage phiPY4 (group III) infected both L. casei and Lactobacillus rhamnosus. Phage phiPY5 (group IV) specifically infected Lactobacillus casei. Morphologically, three phages of groups I belonged to the Myoviridae family, while seven other phages of groups II, III and V belonged to the Siphoviridae family. SDS-PAGE profiles, restriction analysis, G + C contents of DNA and Dot blot hybridization revealed a high degree of homology in each group. Clustering derived from host range analysis was closely related to results of DNA and protein analyses. These phages may be applicable to phage typing for silage-making lactobacilli. PMID:16233449

  4. Molecular imaging of T4 phage in mammalian tissues and cells

    PubMed Central

    Kaźmierczak, Zuzanna; Piotrowicz, Agnieszka; Owczarek, Barbara; Hodyra, Katarzyna; Miernikiewicz, Paulina; Lecion, Dorota; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2014-01-01

    Advances in phage therapy encourage scientific interest in interactions of phages with human and animal organisms. This has created a need for developing tools that facilitate studies of phage circulation and deposition in tissues and cells. Here we propose a new green fluorescent protein (GFP)-based method for T4 phage molecular imaging in living systems. The method employs decoration of a phage capsid with GFP fused to the N-terminus of Hoc protein by in vivo phage display. Fluorescent phages were positively assessed as regards their applicability for detection inside living mammalian cells (by phagocytosis) and tissues (filtering and retention by lymph nodes and spleen). Molecular imaging provides innovative techniques that have brought substantial progress in life sciences. We propose it as a useful tool for studies of phage biology. PMID:24653943

  5. Biochemical functionalization of peptide nanotubes with phage displayed peptides

    NASA Astrophysics Data System (ADS)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

  6. Biochemical functionalization of peptide nanotubes with phage displayed peptides.

    PubMed

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering. PMID:27479451

  7. A simple method for the isolation of phages from Listeria monocytogenes.

    PubMed

    Durst, J; Rau, E; Kemenes, F; Berencsi, G

    1980-02-01

    By application of a combined mitomycin-/heat treatment after freezing, 7 out of 29 Listeria monocytogenes strains which were found to be non-phage carriers by UV irradiation could release phages. Propagation of the obtained phages was promoted by storage at 4 degrees C. Apart from TNSA plates the application of chocolate plates appears to be necessary in order to study these phages.

  8. Phage cluster relationships identified through single gene analysis

    PubMed Central

    2013-01-01

    Background Phylogenetic comparison of bacteriophages requires whole genome approaches such as dotplot analysis, genome pairwise maps, and gene content analysis. Currently mycobacteriophages, a highly studied phage group, are categorized into related clusters based on the comparative analysis of whole genome sequences. With the recent explosion of phage isolation, a simple method for phage cluster prediction would facilitate analysis of crude or complex samples without whole genome isolation and sequencing. The hypothesis of this study was that mycobacteriophage-cluster prediction is possible using comparison of a single, ubiquitous, semi-conserved gene. Tape Measure Protein (TMP) was selected to test the hypothesis because it is typically the longest gene in mycobacteriophage genomes and because regions within the TMP gene are conserved. Results A single gene, TMP, identified the known Mycobacteriophage clusters and subclusters using a Gepard dotplot comparison or a phylogenetic tree constructed from global alignment and maximum likelihood comparisons. Gepard analysis of 247 mycobacteriophage TMP sequences appropriately recovered 98.8% of the subcluster assignments that were made by whole-genome comparison. Subcluster-specific primers within TMP allow for PCR determination of the mycobacteriophage subcluster from DNA samples. Using the single-gene comparison approach for siphovirus coliphages, phage groupings by TMP comparison reflected relationships observed in a whole genome dotplot comparison and confirm the potential utility of this approach to another widely studied group of phages. Conclusions TMP sequence comparison and PCR results support the hypothesis that a single gene can be used for distinguishing phage cluster and subcluster assignments. TMP single-gene analysis can quickly and accurately aid in mycobacteriophage classification. PMID:23777341

  9. The diverse genetic switch of enterobacterial and marine telomere phages.

    PubMed

    Hammerl, Jens A; Jäckel, Claudia; Funk, Eugenia; Pinnau, Sabrina; Mache, Christin; Hertwig, Stefan

    2016-01-01

    Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the enterobacterial telomere phages N15, PY54 and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator, similar to CI, Cro and Q of lambda, respectively. Moreover, N15 and ϕKO2 contain 3 related operator (OR) sites between cI and cro, while only one site (OR3) has been detected in PY54. Marine telomere phages possess a putative cI gene but not a cro-like gene. Instead, a gene is located at the position of cro, whose product shows some similarity to the PY54 ORF42 product, the function of which is unknown. We have determined the transcription start sites of the predicted repressor genes of N15, PY54, ϕKO2 and of the marine telomere phage VP58.5. The influence of the genes on phage propagation was analyzed in E. coli, Y. enterocolitica and V.parahaemolyticus. We show that the repressors and antiterminators of N15, ϕKO2 and PY54 exerted their predicted activities. However, while the proteins of both N15 and ϕKO2 affected lysis and lysogeny by N15, they did not affect PY54 propagation. On the other hand, the respective PY54 proteins exclusively influenced the propagation of this phage. The immB region of VP58.5 contains 2 genes that revealed prophage repressor activity, while a lytic repressor gene could not be identified. The results indicate an unexpected diversity of the growth regulation mechanisms in these temperate phages.

  10. The diverse genetic switch of enterobacterial and marine telomere phages.

    PubMed

    Hammerl, Jens A; Jäckel, Claudia; Funk, Eugenia; Pinnau, Sabrina; Mache, Christin; Hertwig, Stefan

    2016-01-01

    Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the enterobacterial telomere phages N15, PY54 and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator, similar to CI, Cro and Q of lambda, respectively. Moreover, N15 and ϕKO2 contain 3 related operator (OR) sites between cI and cro, while only one site (OR3) has been detected in PY54. Marine telomere phages possess a putative cI gene but not a cro-like gene. Instead, a gene is located at the position of cro, whose product shows some similarity to the PY54 ORF42 product, the function of which is unknown. We have determined the transcription start sites of the predicted repressor genes of N15, PY54, ϕKO2 and of the marine telomere phage VP58.5. The influence of the genes on phage propagation was analyzed in E. coli, Y. enterocolitica and V.parahaemolyticus. We show that the repressors and antiterminators of N15, ϕKO2 and PY54 exerted their predicted activities. However, while the proteins of both N15 and ϕKO2 affected lysis and lysogeny by N15, they did not affect PY54 propagation. On the other hand, the respective PY54 proteins exclusively influenced the propagation of this phage. The immB region of VP58.5 contains 2 genes that revealed prophage repressor activity, while a lytic repressor gene could not be identified. The results indicate an unexpected diversity of the growth regulation mechanisms in these temperate phages. PMID:27607141

  11. Genomes and Characterization of Phages Bcep22 and BcepIL02, Founders of a Novel Phage Type in Burkholderia cenocepacia▿†

    PubMed Central

    Gill, Jason J.; Summer, Elizabeth J.; Russell, William K.; Cologna, Stephanie M.; Carlile, Thomas M.; Fuller, Alicia C.; Kitsopoulos, Kate; Mebane, Leslie M.; Parkinson, Brandi N.; Sullivan, David; Carmody, Lisa A.; Gonzalez, Carlos F.; LiPuma, John J.; Young, Ry

    2011-01-01

    Within the Burkholderia cepacia complex, B. cenocepacia is the most common species associated with aggressive infections in the lungs of cystic fibrosis patients, causing disease that is often refractive to treatment by antibiotics. Phage therapy may be a potential alternative form of treatment for these infections. Here we describe the genome of the previously described therapeutic B. cenocepacia podophage BcepIL02 and its close relative, Bcep22. Phage Bcep22 was found to contain a circularly permuted genome of 63,882 bp containing 77 genes; BcepIL02 was found to be 62,714 bp and contains 76 predicted genes. Major virion-associated proteins were identified by proteomic analysis. We propose that these phages comprise the founding members of a novel podophage lineage, the Bcep22-like phages. Among the interesting features of these phages are a series of tandemly repeated putative tail fiber genes that are similar to each other and also to one or more such genes in the other phages. Both phages also contain an extremely large (ca. 4,600-amino-acid), virion-associated, multidomain protein that accounts for over 20% of the phages' coding capacity, is widely distributed among other bacterial and phage genomes, and may be involved in facilitating DNA entry in both phage and other mobile DNA elements. The phages, which were previously presumed to be virulent, show evidence of a temperate lifestyle but are apparently unable to form stable lysogens in their hosts. This ambiguity complicates determination of a phage lifestyle, a key consideration in the selection of therapeutic phages. PMID:21804006

  12. Genome Sequence of Bacillus cereus Phage vB_BceS-MY192.

    PubMed

    Yang, Yong; Zhan, Li; Chen, Jiancai; Zhang, Yunyi; Sun, Yi; Yang, Zhangnv; Jiang, Liping; Zhu, Hanping; Zhang, Yanjun; Lu, Yiyu; Mei, Lingling

    2016-01-01

    ITALIC! Bacillus cereusis an opportunistic foodborne pathogen. The phage vB_BceS-MY192 was isolated from ITALIC! B. cereus192 in a cooked rice sample. The temperate phage belongs to the ITALIC! Siphoviridaefamily, ITALIC! Caudoviralesorder. Here we announce the phage genome sequence and its annotation, which may expand the understanding of ITALIC! B. cereussiphophages.

  13. Genome Sequences of Gordonia Phages BaxterFox, Kita, Nymphadora, and Yeezy

    PubMed Central

    Bandla, Sharanya; Colbert, Alexandra K.; Eichinger, Fiona G.; Gamburg, Michelle B.; Horiates, Stavroula G.; Jamison, Jerrica M.; Julian, Dana R.; Moore, Whitney A.; Murthy, Pranav; Powell, Meghan C.; Smith, Sydney V.; Mezghani, Nadia; Milliken, Katherine A.; Thompson, Paige K.; Toner, Chelsea L.; Ulbrich, Megan C.; 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

    Gordonia phages BaxterFox, Kita, Nymphadora, and Yeezy are newly characterized phages of Gordonia terrae, isolated from soil samples in Pittsburgh, Pennsylvania. These phages have genome lengths between 50,346 and 53,717 bp, and encode on average 84 predicted proteins. All have G+C content of 66.6%. PMID:27516501

  14. Genome Sequence of Bacillus cereus Phage vB_BceS-MY192

    PubMed Central

    Yang, Yong; Zhan, Li; Chen, Jiancai; Zhang, Yunyi; Sun, Yi; Yang, Zhangnv; Jiang, Liping; Zhu, Hanping; Zhang, Yanjun; Lu, Yiyu

    2016-01-01

    Bacillus cereus is an opportunistic foodborne pathogen. The phage vB_BceS-MY192 was isolated from B. cereus 192 in a cooked rice sample. The temperate phage belongs to the Siphoviridae family, Caudovirales order. Here we announce the phage genome sequence and its annotation, which may expand the understanding of B. cereus siphophages. PMID:27103733

  15. Phage mutations in response to CRISPR diversification in a bacterial population.

    PubMed

    Sun, Christine L; Barrangou, Rodolphe; Thomas, Brian C; Horvath, Philippe; Fremaux, Christophe; Banfield, Jillian F

    2013-02-01

    Interactions between bacteria and their coexisting phage populations impact evolution and can strongly influence biogeochemical processes in natural ecosystems. Periodically, mutation or migration results in exposure of a host to a phage to which it has no immunity; alternatively, a phage may be exposed to a host it cannot infect. To explore the processes by which coexisting, co-evolving hosts and phage populations establish, we cultured Streptococcus thermophilus DGCC7710 with phage 2972 and tracked CRISPR (clustered regularly interspaced short palindromic repeats) diversification and host-phage co-evolution in a population derived from a colony that acquired initial CRISPR-encoded immunity. After 1 week of co-culturing, the coexisting host-phage populations were metagenomically characterized using 454 FLX Titanium sequencing. The evolved genomes were compared with reference genomes to identify newly incorporated spacers in S. thermophilus DGCC7710 and recently acquired single-nucleotide polymorphisms (SNPs) in phage 2972. Following phage exposure, acquisition of immune elements (spacers) led to a genetically diverse population with multiple subdominant strain lineages. Phage mutations that circumvented three early immunization events were localized in the proto-spacer adjacent motif (PAM) or near the PAM end of the proto-spacer, suggesting a strong selective advantage for the phage that mutated in this region. The sequential fixation or near fixation of these single mutations indicates selection events so severe that single phage genotypes ultimately gave rise to all surviving lineages and potentially carried traits unrelated to immunity to fixation.

  16. Multidimensional metrics for estimating phage abundance, distribution, gene density, and sequence coverage in metagenomes

    DOE PAGES

    Aziz, Ramy K.; Dwivedi, Bhakti; Akhter, Sajia; Breitbart, Mya; Edwards, Robert A.

    2015-05-08

    Phages are the most abundant biological entities on Earth and play major ecological roles, yet the current sequenced phage genomes do not adequately represent their diversity, and little is known about the abundance and distribution of these sequenced genomes in nature. Although the study of phage ecology has benefited tremendously from the emergence of metagenomic sequencing, a systematic survey of phage genes and genomes in various ecosystems is still lacking, and fundamental questions about phage biology, lifestyle, and ecology remain unanswered. To address these questions and improve comparative analysis of phages in different metagenomes, we screened a core set ofmore » publicly available metagenomic samples for sequences related to completely sequenced phages using the web tool, Phage Eco-Locator. We then adopted and deployed an array of mathematical and statistical metrics for a multidimensional estimation of the abundance and distribution of phage genes and genomes in various ecosystems. Experiments using those metrics individually showed their usefulness in emphasizing the pervasive, yet uneven, distribution of known phage sequences in environmental metagenomes. Using these metrics in combination allowed us to resolve phage genomes into clusters that correlated with their genotypes and taxonomic classes as well as their ecological properties. By adding this set of metrics to current metaviromic analysis pipelines, where they can provide insight regarding phage mosaicism, habitat specificity, and evolution.« less

  17. Multidimensional metrics for estimating phage abundance, distribution, gene density, and sequence coverage in metagenomes

    SciTech Connect

    Aziz, Ramy K.; Dwivedi, Bhakti; Akhter, Sajia; Breitbart, Mya; Edwards, Robert A.

    2015-05-08

    Phages are the most abundant biological entities on Earth and play major ecological roles, yet the current sequenced phage genomes do not adequately represent their diversity, and little is known about the abundance and distribution of these sequenced genomes in nature. Although the study of phage ecology has benefited tremendously from the emergence of metagenomic sequencing, a systematic survey of phage genes and genomes in various ecosystems is still lacking, and fundamental questions about phage biology, lifestyle, and ecology remain unanswered. To address these questions and improve comparative analysis of phages in different metagenomes, we screened a core set of publicly available metagenomic samples for sequences related to completely sequenced phages using the web tool, Phage Eco-Locator. We then adopted and deployed an array of mathematical and statistical metrics for a multidimensional estimation of the abundance and distribution of phage genes and genomes in various ecosystems. Experiments using those metrics individually showed their usefulness in emphasizing the pervasive, yet uneven, distribution of known phage sequences in environmental metagenomes. Using these metrics in combination allowed us to resolve phage genomes into clusters that correlated with their genotypes and taxonomic classes as well as their ecological properties. By adding this set of metrics to current metaviromic analysis pipelines, where they can provide insight regarding phage mosaicism, habitat specificity, and evolution.

  18. Phage mutations in response to CRISPR diversification in a bacterial population.

    PubMed

    Sun, Christine L; Barrangou, Rodolphe; Thomas, Brian C; Horvath, Philippe; Fremaux, Christophe; Banfield, Jillian F

    2013-02-01

    Interactions between bacteria and their coexisting phage populations impact evolution and can strongly influence biogeochemical processes in natural ecosystems. Periodically, mutation or migration results in exposure of a host to a phage to which it has no immunity; alternatively, a phage may be exposed to a host it cannot infect. To explore the processes by which coexisting, co-evolving hosts and phage populations establish, we cultured Streptococcus thermophilus DGCC7710 with phage 2972 and tracked CRISPR (clustered regularly interspaced short palindromic repeats) diversification and host-phage co-evolution in a population derived from a colony that acquired initial CRISPR-encoded immunity. After 1 week of co-culturing, the coexisting host-phage populations were metagenomically characterized using 454 FLX Titanium sequencing. The evolved genomes were compared with reference genomes to identify newly incorporated spacers in S. thermophilus DGCC7710 and recently acquired single-nucleotide polymorphisms (SNPs) in phage 2972. Following phage exposure, acquisition of immune elements (spacers) led to a genetically diverse population with multiple subdominant strain lineages. Phage mutations that circumvented three early immunization events were localized in the proto-spacer adjacent motif (PAM) or near the PAM end of the proto-spacer, suggesting a strong selective advantage for the phage that mutated in this region. The sequential fixation or near fixation of these single mutations indicates selection events so severe that single phage genotypes ultimately gave rise to all surviving lineages and potentially carried traits unrelated to immunity to fixation. PMID:23057534

  19. Genome Sequences of Gordonia Phages BaxterFox, Kita, Nymphadora, and Yeezy.

    PubMed

    Pope, Welkin H; Bandla, Sharanya; Colbert, Alexandra K; Eichinger, Fiona G; Gamburg, Michelle B; Horiates, Stavroula G; Jamison, Jerrica M; Julian, Dana R; Moore, Whitney A; Murthy, Pranav; Powell, Meghan C; Smith, Sydney V; Mezghani, Nadia; Milliken, Katherine A; Thompson, Paige K; Toner, Chelsea L; Ulbrich, Megan C; 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

    Gordonia phages BaxterFox, Kita, Nymphadora, and Yeezy are newly characterized phages of Gordonia terrae, isolated from soil samples in Pittsburgh, Pennsylvania. These phages have genome lengths between 50,346 and 53,717 bp, and encode on average 84 predicted proteins. All have G+C content of 66.6%. PMID:27516501

  20. Multidimensional metrics for estimating phage abundance, distribution, gene density, and sequence coverage in metagenomes.

    PubMed

    Aziz, Ramy K; Dwivedi, Bhakti; Akhter, Sajia; Breitbart, Mya; Edwards, Robert A

    2015-01-01

    Phages are the most abundant biological entities on Earth and play major ecological roles, yet the current sequenced phage genomes do not adequately represent their diversity, and little is known about the abundance and distribution of these sequenced genomes in nature. Although the study of phage ecology has benefited tremendously from the emergence of metagenomic sequencing, a systematic survey of phage genes and genomes in various ecosystems is still lacking, and fundamental questions about phage biology, lifestyle, and ecology remain unanswered. To address these questions and improve comparative analysis of phages in different metagenomes, we screened a core set of publicly available metagenomic samples for sequences related to completely sequenced phages using the web tool, Phage Eco-Locator. We then adopted and deployed an array of mathematical and statistical metrics for a multidimensional estimation of the abundance and distribution of phage genes and genomes in various ecosystems. Experiments using those metrics individually showed their usefulness in emphasizing the pervasive, yet uneven, distribution of known phage sequences in environmental metagenomes. Using these metrics in combination allowed us to resolve phage genomes into clusters that correlated with their genotypes and taxonomic classes as well as their ecological properties. We propose adding this set of metrics to current metaviromic analysis pipelines, where they can provide insight regarding phage mosaicism, habitat specificity, and evolution.

  1. Selective posttranslational modification of phage-displayed polypeptides

    DOEpatents

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-02-05

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2]cycloaddition reactions and Staudinger modifications.

  2. Selective posttranslational modification of phage-displayed polypeptides

    DOEpatents

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-11-19

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2] cycloaddition reactions and Staudinger modifications.

  3. Genetics of the phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2).

    PubMed

    Sumby, Paul; Smith, Margaret C M

    2002-04-01

    The phage growth limitation (Pgl) system, encoded by Streptomyces coelicolor A3(2), confers protection against the temperate bacteriophage phiC31 and its homoimmune relatives. The Pgl phenotype is characterized by the ability of Pgl+ hosts to support a phage burst on initial infection but subsequent cycles are severely attenuated. Previously, two adjacent genes pglY and pglZ were shown to be required for Pgl. It had been shown by Southern blotting that Streptomyces lividans, a close relative of S. coelicolor and naturally Pgl-, does not contain homologues of pglYZ and that introduction of pglYZ into S. lividans is not sufficient to confer a Pgl+ phenotype. Moreover, the mechanism of the Pgl+<--> Pgl- phase variation associated with this phenotype is also not understood. Here we describe two novel genes, pglW and pglX, that were shown to be part of this system by complementation of Pgl- mutants and by insertional mutagenesis. pglW encodes a 169 kDa protein that includes putative motifs for both serine/threonine protein kinase activity and DNA binding. pglX encodes a 136 kDa protein with putative adenine-specific DNA methyltransferase activity. pglW and pglX have overlapping stop-start codons suggesting transcriptional and translational coupling. S1 mapping of transcripts initiating up-stream of pglW indicated that, like pglYZ, pglWX is expressed in uninfected cultures. A homologue of pglX with 76% amino acid identity was identified in S. coelicolor, and insertional mutagenesis indicated that this gene was not required for the Pgl+ phenotype. Southern blots indicated that S. lividans does not contain homologues of pglW or pglX. A plasmid encoding pglWXYZ was able to confer the Pgl+ phenotype to S. lividans implying that these four genes constitute the whole system.

  4. Rapid Detection of Bacillus anthracis in Complex Food Matrices Using Phage-Mediated Bioluminescence.

    PubMed

    Sharp, Natasha J; Vandamm, Joshua P; Molineux, Ian J; Schofield, David A

    2015-05-01

    Bacillus anthracis, the causative agent of anthrax, is considered a high-priority agent that may be used in a food-related terrorist attack because it can be contracted by ingestion and it also forms spores with heat and chemical resistance. Thus, novel surveillance methodologies to detect B. anthracis on adulterated foods are important for bioterrorism preparedness. We describe the development of a phage-based bioluminescence assay for the detection of B. anthracis on deliberately contaminated foods. We previously engineered the B. anthracis phage Wβ with genes encoding bacterial luciferase (luxA and luxB) to create a "light-tagged" reporter (Wβ::luxAB) that is able to rapidly detect B. anthracis by transducing a bioluminescent signal response. Here, we investigate the ability of Wβ::luxAB to detect B. anthracis Sterne, an attenuated select agent strain, in inoculated food (ground beef) and milk (2%, baby formula, and half and half) matrices after incubation with spores for 72 h at 4°C as per AOAC testing guidelines. The majority of B. anthracis bacilli remained in spore form, and thus were potentially infectious, within each of the liquid matrices for 14 days. Detection limits were 80 CFU/ml after 7 h of enrichment; sensitivity of detection increased to 8 CFU/ml when enrichment was extended to 16 h. The limit of detection in ground beef was 3.2 × 10(3) CFU/g after 7 h of enrichment, improving to 3.2 × 10(2) CFU/g after 16 h. Because the time to result is rapid and minimal processing is required, and because gastrointestinal anthrax can be fatal, the reporter technology displays promise for the protection of our food supply following a deliberate release of this priority pathogen.

  5. Variable laser attenuator

    DOEpatents

    Foltyn, S.R.

    1987-05-29

    The disclosure relates to low loss, high power variable attenuators comprising one or more transmissive and/or reflective multilayer dielectric filters. The attenuator is particularly suitable to use with unpolarized lasers such as excimer lasers. Beam attenuation is a function of beam polarization and the angle of incidence between the beam and the filter and is controlled by adjusting the angle of incidence the beam makes to the filter or filters. Filters are selected in accordance with beam wavelength. 9 figs.

  6. Variable laser attenuator

    DOEpatents

    Foltyn, Stephen R.

    1988-01-01

    The disclosure relates to low loss, high power variable attenuators comprng one or more transmissive and/or reflective multilayer dielectric filters. The attenuator is particularly suitable to use with unpolarized lasers such as excimer lasers. Beam attenuation is a function of beam polarization and the angle of incidence between the beam and the filter and is controlled by adjusting the angle of incidence the beam makes to the filter or filters. Filters are selected in accordance with beam wavelength.

  7. Elevated lytic phage production as a consequence of particle colonization by a marine Flavobacterium (Cellulophaga sp.).

    PubMed

    Riemann, Lasse; Grossart, Hans-Peter

    2008-10-01

    Bacteria growing on marine particles generally have higher densities and cell-specific activities than free-living bacteria. Since rapidity of phage adsorption is dependent on host density, while infection productivity is a function of host physiological status, we hypothesized that marine particles are sites of elevated phage production. In the present study, organic-matter-rich agarose beads and a marine phage-host pair (Cellulophaga sp., PhiS(M)) were used as a model system to examine whether bacterial colonization of particles increases phage production. While no production of phages was observed in plain seawater, the presence of beads enhanced attachment and growth of bacteria, as well as phage production. This was observed because of extensive lysis of bacteria in the presence of beads and a subsequent increase in phage abundance both on beads and in the surrounding water. After 12 h, extensive phage lysis reduced the density of attached bacteria; however, after 32 h, bacterial abundance increased again. Reexposure to phages and analyses of bacterial isolates suggested that this regrowth on particles was by phage-resistant clones. The present demonstration of elevated lytic phage production associated with model particles illustrates not only that a marine phage has the ability to successfully infect and lyse surface-attached bacteria but also that acquisition of resistance may affect temporal phage-host dynamics on particles. These findings from a model system may have relevance to the distribution of phage production in environments rich in particulate matter (e.g., in coastal areas or during phytoplankton blooms) where a significant part of phage production may be directly linked to these nutrient-rich "hot spots."

  8. Prevalence of Stx phages in environments of a pig farm and lysogenic infection of the field E. coli O157 isolates with a recombinant converting Phage.

    PubMed

    Yan, Yaxian; Shi, Yibo; Cao, Dongmei; Meng, Xiangpeng; Xia, Luming; Sun, Jianhe

    2011-02-01

    The prevalence and nature of Shiga toxin (Stx)-producing Escherichia coli (STEC) and Stx phage were investigated in 720 swine fecal samples randomly collected from a commercial breeding pig farm in China over a 1-year surveillance period. Eight STEC O157 (1.1%), 33 STEC non-O157 (4.6%), and two stx-negative O157 (0.3%) isolates were identified. Fecal filtrates were screened directly for Stx phages using E. coli K-12 derivative strains MC1061 as indicator, yielding 15 Stx1 and 57 Stx2 phages. One Stx1 and eight Stx2 phages were obtained following norfloxacin induction of the eight field STEC O157 isolates. All Stx1 phages had hexagonal heads with long tails, while Stx2 phages had three different morphologies. Notably, most of field STEC O157 isolates released more free phages and Stx toxin after induction with ciprofloxacin. Furthermore, upon infection with the recombinant phage ΦMin27(Δstx::cat), E. coli laboratory strains produced both lysogenic and lytic phage, whereas two of the eight O157 STEC isolates produced only lysogens. The lysogens from laboratory strains produced infectious particles similar to ΦMin27. Similarly, the lysogens from the STEC O157 isolates released Stx phage too, although free ΦMin27(Δstx::cat) particles were not detected. Collectively, our results reveal that breeding pig farms could be important reservoirs for Stx phages and that residual antibacterial agents may enhance the release of Stx phages and the expression of Stx. PMID:20697714

  9. Prevalence of Stx phages in environments of a pig farm and lysogenic infection of the field E. coli O157 isolates with a recombinant converting Phage.

    PubMed

    Yan, Yaxian; Shi, Yibo; Cao, Dongmei; Meng, Xiangpeng; Xia, Luming; Sun, Jianhe

    2011-02-01

    The prevalence and nature of Shiga toxin (Stx)-producing Escherichia coli (STEC) and Stx phage were investigated in 720 swine fecal samples randomly collected from a commercial breeding pig farm in China over a 1-year surveillance period. Eight STEC O157 (1.1%), 33 STEC non-O157 (4.6%), and two stx-negative O157 (0.3%) isolates were identified. Fecal filtrates were screened directly for Stx phages using E. coli K-12 derivative strains MC1061 as indicator, yielding 15 Stx1 and 57 Stx2 phages. One Stx1 and eight Stx2 phages were obtained following norfloxacin induction of the eight field STEC O157 isolates. All Stx1 phages had hexagonal heads with long tails, while Stx2 phages had three different morphologies. Notably, most of field STEC O157 isolates released more free phages and Stx toxin after induction with ciprofloxacin. Furthermore, upon infection with the recombinant phage ΦMin27(Δstx::cat), E. coli laboratory strains produced both lysogenic and lytic phage, whereas two of the eight O157 STEC isolates produced only lysogens. The lysogens from laboratory strains produced infectious particles similar to ΦMin27. Similarly, the lysogens from the STEC O157 isolates released Stx phage too, although free ΦMin27(Δstx::cat) particles were not detected. Collectively, our results reveal that breeding pig farms could be important reservoirs for Stx phages and that residual antibacterial agents may enhance the release of Stx phages and the expression of Stx.