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Sample records for broad range 16s

  1. Diagnosis of neonatal sepsis by broad-range 16S real-time polymerase chain reaction.

    PubMed

    Ohlin, Andreas; Bäckman, Anders; Ewald, Uwe; Schollin, Jens; Björkqvist, Maria

    2012-01-01

    The standard diagnostic test (blood culture) for suspected neonatal sepsis has limitations in sensitivity and specificity, and 16S polymerase chain reaction (PCR) has been suggested as a new diagnostic tool for neonatal sepsis. To develop and evaluate a new real-time PCR method for detection of bacterial DNA in blood samples collected from infants with suspected neonatal sepsis. Immediately after blood culture, a study sample of 0.5-1.0 ml whole blood was collected and used for a novel 16S real-time PCR assay. All positive samples were sequenced. Detailed case studies were performed in all cases with conflicting results, to verify if PCR could detect pathogens in culture negative sepsis. 368 samples from 317 infants were included. When compared with blood culture, the assay yielded a sensitivity of 79%, a specificity of 90%, a positive predictive value of 59%, and a negative predictive value of 96%. Seven of the 31 samples with a positive PCR result and a negative blood culture had definite or suspected bacterial sepsis. In five samples, PCR (but not blood culture) could detect a pathogen that was present in a blood culture collected more than 24 h prior to the PCR sample. This study presents an evaluation of a new real-time PCR technique that can detect culture-positive sepsis, and suggests that PCR has the potential to detect bacteria in culture-negative samples even after the initiation of intravenous antibiotics. Copyright © 2011 S. Karger AG, Basel.

  2. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

    DOE PAGES

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; ...

    2015-02-06

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n =more » 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.« less

  3. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

    SciTech Connect

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.; Heimesaat, Markus M.

    2015-02-06

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.

  4. Use of 16S rRNA Gene for Identification of a Broad Range of Clinically Relevant Bacterial Pathogens

    PubMed Central

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.

    2015-01-01

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci. PMID:25658760

  5. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens.

    PubMed

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L; Lynch, Susan V

    2015-01-01

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.

  6. [A case of culture-negative brain abscess caused by Streptococcus intermedius infection diagnosed by broad-range PCR of 16S ribosomal RNA].

    PubMed

    Ohara, Nobuyuki; Asai, Katsunori; Ohkusu, Kiyofumi; Wakayama, Akatsuki

    2013-10-01

    A 50-year-old man presented with altered mental status during hospitalization for pneumonia. MRI showed multifocal ring-enhanced lesions, which consisted of multiple cerebral abscesses. We started empirical antibiotic therapy, but the following morning, his condition rapidly deteriorated and a CT scan revealed acute hydrocephalus, which required ventricular drainage. Gram staining of cerebro-spinal fluid from the ventricular drainage showed gram-positive cocci in chains, but culture results were negative. 16S ribosomal RNA sequencing with broad-range PCR of the cerebro-spinal fluid identified Streptococcus intermedius. On the basis of this identification, the antibiotic regimen was changed to ampicillin monotherapy. After 1 year of antibiotic therapy, all the abscesses had disappeared and the patient was discharged without any sequelae. Bacterial 16S rRNA gene analysis with broad-range PCR is a very useful method for facilitating the etiological diagnosis and selection of appropriate treatment for culture-negative infections.

  7. Development of a broad-range 16S rDNA real-time PCR for the diagnosis of septic arthritis in children.

    PubMed

    Rosey, Anne-Laure; Abachin, Eric; Quesnes, Gilles; Cadilhac, Céline; Pejin, Zagorka; Glorion, Christophe; Berche, Patrick; Ferroni, Agnès

    2007-01-01

    The broad-range PCR has been successfully developed to search for fastidious, slow-growing or uncultured bacteria, and is mostly used when an empirical antibiotic treatment has already been initiated. The technique generally involves standard PCR targeting the gene coding for 16S ribosomal RNA, and includes a post-PCR visualisation step on agarose gel which is a potential source of cross-over contamination. In addition, interpretation of the presence of amplified products on gels can be difficult. We then developed a new SYBR Green-based, universal real-time PCR assay targeting the gene coding for 16S ribosomal RNA, coupled with sequencing of amplified products. The real-time PCR assay was evaluated on 94 articular fluid samples collected from children hospitalised for suspicion of septic arthritis, as compared to the results obtained with bacterial cultures and conventional broad-range PCR. DNA extraction was performed with the automated MagNa Pure system. We could detect DNA from various bacterial pathogens including fastidious bacteria (Kingella kingae, Streptococcus pneumoniae, Streptococcus pyogenes, Salmonella spp, Staphylococcus aureus) from 23% of cases of septic arthritis giving negative culture results. The real-time technique was easier to interpret and allowed to detect four more cases than conventional PCR. PCR based molecular techniques appear to be essential to perform in case of suspicion of septic arthritis, provided the increase of the diagnosed bacterial etiologies. Real-time PCR technique is a sensitive and reliable technique, which can replace conventional PCR for clinical specimens with negative bacterial culture.

  8. Broad-range 16S rRNA PCR with cerebrospinal fluid may be unreliable for management of postoperative aseptic meningitis.

    PubMed

    Zarrouk, Virginie; Leflon-Guibout, Véronique; Robineaux, Sébastien; Kalamarides, Michel; Nicolas-Chanoine, Marie-Hélène; Sterkers, Olivier; Fantin, Bruno

    2010-09-01

    We previously demonstrated that discontinuing presumptive antibiotic treatment in cases of negative conventional cultures is safe and effective for patients with postoperative aseptic meningitis (PAM). Here, we prospectively investigated 32 patients with postoperative meningitis. All 26 patients with PAM diagnosed on the basis of conventional cultures demonstrated negative 16S rRNA PCR results. Our results suggest that the PCR technique does not change PAM management.

  9. Identification of bacterial pathogens in ascitic fluids from patients with suspected spontaneous bacterial peritonitis by use of broad-range PCR (16S PCR) coupled with high-resolution melt analysis.

    PubMed

    Hardick, Justin; Won, Helen; Jeng, Kevin; Hsieh, Yu-Hsiang; Gaydos, Charlotte A; Rothman, Richard E; Yang, Samuel

    2012-07-01

    Spontaneous bacterial peritonitis (SBP) can be a severe complication occurring in patients with cirrhosis and ascites, with associated mortality often as high as 40%. Traditional diagnostics for SBP rely on culture techniques for proper diagnosis, although recent reports suggest that the presence of bacterial DNA in peritoneal fluid in patients with cirrhosis and ascites is an indicator of SBP. A previously published broad-range PCR (16S PCR) coupled with high-resolution melt analysis (HRMA) was compared with standard culture techniques for diagnosis of SBP in 106 peritoneal fluid samples from patients with suspected SBP. The sensitivity and specificity for 16S PCR for detecting eubacterial DNA compared with those of standard culture techniques were 100% (17/17) and 91.5% (85/89), respectively. Overall, HRMA concordance with species identification was 70.6% (12/17), although the 5 samples that were discordant at the species level were SBP resulting from a polymicrobial infection, and species-level identification for polymicrobial infections is outside the capability of HRMA. Both the broad-range 16S PCR and HRMA analysis provide useful diagnostic adjunctive assays for clinicians in detecting and identifying pathogens responsible for SBP.

  10. Broad-range PCR, cloning and sequencing of the full 16S rRNA gene for detection of bacterial DNA in synovial fluid samples of Tunisian patients with reactive and undifferentiated arthritis

    PubMed Central

    Siala, Mariam; Gdoura, Radhouane; Fourati, Hela; Rihl, Markus; Jaulhac, Benoit; Younes, Mohamed; Sibilia, Jean; Baklouti, Sofien; Bargaoui, Naceur; Sellami, Slaheddine; Sghir, Abdelghani; Hammami, Adnane

    2009-01-01

    Introduction Broad-range rDNA PCR provides an alternative, cultivation-independent approach for identifying bacterial DNA in reactive and other form of arthritis. The aim of this study was to use broad-range rDNA PCR targeting the 16S rRNA gene in patients with reactive and other forms of arthritis and to screen for the presence of DNA from any given bacterial species in synovial fluid (SF) samples. Methods We examined the SF samples from a total of 27 patients consisting of patients with reactive arthritis (ReA) (n = 5), undifferentiated arthritis (UA) (n = 9), rheumatoid arthritis (n = 7), and osteoarthritis (n = 6) of which the latter two were used as controls. Using broad-range bacterial PCR amplifying a 1400 bp fragment from the 16S rRNA gene, we identified and sequenced at least 24 clones from each SF sample. To identify the corresponding bacteria, DNA sequences were compared to the EMBL (European Molecular Biology Laboratory) database. Results Bacterial DNA was identified in 20 of the 27 SF samples (74, 10%). Analysis of a large number of sequences revealed the presence of DNA from more than one single bacterial species in the SF of all patients studied. The nearly complete sequences of the 1400 bp were obtained for most of the detected species. DNA of bacterial species including Shigella species, Escherichia species, and other coli-form bacteria as well as opportunistic pathogens such as Stenotrophomonas maltophilia and Achromobacter xylosoxidans were shared in all arthritis patients. Among pathogens described to trigger ReA, DNA from Shigella sonnei was found in ReA and UA patients. We also detected DNA from rarely occurring human pathogens such as Aranicola species and Pantoea ananatis. We also found DNA from bacteria so far not described in human infections such as Bacillus niacini, Paenibacillus humicus, Diaphorobacter species and uncultured bacterium genera incertae sedis OP10. Conclusions Broad-range PCR followed by cloning and sequencing the entire

  11. Broad host range plasmids.

    PubMed

    Jain, Aayushi; Srivastava, Preeti

    2013-11-01

    Plasmids are and will remain important cloning vehicles for biotechnology. They have also been associated with the spread of a number of diseases and therefore are a subject of environmental concern. With the advent of sequencing technologies, the database of plasmids is increasing. It will be of immense importance to identify the various bacterial hosts in which the plasmid can replicate. The present review article describes the features that confer broad host range to the plasmids, the molecular basis of plasmid host range evolution, and applications in recombinant DNA technology and environment.

  12. Broad-range real time PCR and DNA sequencing for the diagnosis of bacterial meningitis.

    PubMed

    Deutch, Susanna; Pedersen, Lisbeth N; Pødenphant, Lone; Olesen, Rikke; Schmidt, Michael B; Møller, Jens K; Ostergaard, Lars

    2006-01-01

    Rapid aetiological diagnosis of bacterial meningitis is crucial for the early targeting of antimicrobial and adjuvant therapy. Broad-range polymerase chain reaction (PCR) targeting the 16S rRNA gene allows aetiological diagnosis of bacterial meningitis when applied to cerebrospinal fluid (CSF). We assessed the additional diagnostic effect of applying a novel broad-range real time PCR and subsequent DNA sequencing to culture, microscopy, and broad-range conventional PCR on CSF in patients with suspected bacterial meningitis. Broad-range conventional PCR and broad-range real time PCR with subsequent DNA sequencing were applied to 206 CSF specimens collected consecutively from 203 patients aged 6 d to 86 y. Patients' charts were reviewed for clinical information. 17 pathogens were identified by PCR and DNA sequencing or culture. Three specimens were negative by culture but positive by broad-range real time PCR. Three specimens were positive by culture but negative by broad-range real time PCR. Compared with culture, the sensitivity of broad-range real time PCR was 86%, and the specificity 98%. Conventional PCR resulted in a sensitivity of 64% and specificity of 98%. Broad-range real time PCR was generally comparable to culture of CSF and may be a useful supplement, particularly when antimicrobial therapy has been administered. Broad-range real time PCR was more sensitive than broad-range conventional PCR and microscopy.

  13. Development of a novel long-range 16S rRNA universal primer set for metagenomic analysis of gastrointestinal microbiota in newborn infants.

    PubMed

    Ku, Hye-Jin; Lee, Ju-Hoon

    2014-06-28

    Metagenomic analysis of the human intestinal microbiota has extended our understanding of the role of these bacteria in improving human intestinal health; however, a number of reports have shown that current total fecal DNA extraction methods and 16S rRNA universal primer sets could affect the species coverage and resolution of these analyses. Here, we improved the extraction method for total DNA from human fecal samples by optimization of the lysis buffer, boiling time (10 min), and bead-beating time (0 min). In addition, we developed a new longrange 16S rRNA universal PCR primer set targeting the V6 to V9 regions with a 580 bp DNA product length. This new 16S rRNA primer set was evaluated by comparison with two previously developed 16S rRNA universal primer sets and showed high species coverage and resolution. The optimized total fecal DNA extraction method and newly designed long-range 16S rRNA universal primer set will be useful for the highly accurate metagenomic analysis of adult and infant intestinal microbiota with minimization of any bias.

  14. Fluorescence-based Broad Dynamic Range Viscosity Probes.

    PubMed

    Dragan, Anatoliy; Graham, August E; Geddes, Chris D

    2014-03-01

    We introduce two new fluorescent viscosity probes, SYBR Green (SG) and PicoGreen (PG), that we have studied over a broad range of viscosity and in collagen solutions. In water, both dyes have low quantum yields and excited state lifetimes, while in viscous solvents or in complex with DNA both parameters dramatically (300-1000-fold) increase. We show that in log-log scale the dependence of the dyes' quantum yield vs. viscosity is linear, the slope of which is sensitive to temperature. Application of SG and PG, as a fluorescence-based broad dynamic range viscosity probes, to the life sciences is discussed.

  15. More Is Better: Selecting for Broad Host Range Bacteriophages

    PubMed Central

    Ross, Alexa; Ward, Samantha; Hyman, Paul

    2016-01-01

    Bacteriophages are viruses that infect bacteria. In this perspective, we discuss several aspects of a characteristic feature of bacteriophages, their host range. Each phage has its own particular host range, the range of bacteria that it can infect. While some phages can only infect one or a few bacterial strains, other phages can infect many species or even bacteria from different genera. Different methods for determining host range may give different results, reflecting the multiple mechanisms bacteria have to resist phage infection and reflecting the different steps of infection each method depends on. This makes defining host range difficult. Another difficulty in describing host range arises from the inconsistent use of the words “narrow” and especially “broad” when describing the breadth of the host range. Nearly all bacteriophages have been isolated using a single host strain of bacteria. While this procedure is fairly standard, it may more likely produce narrow rather than broad host range phage. Our results and those of others suggest that using multiple host strains during isolation can more reliably produce broader host range phages. This challenges the common belief that most bacteriophages have a narrow host range. We highlight the implications of this for several areas that are affected by host range including horizontal gene transfer and phage therapy. PMID:27660623

  16. New focal plane detector system for the broad range spectrometer

    SciTech Connect

    Sjoreen, T.P.

    1984-01-01

    A focal plane detector system consisting of a vertical drift chamber, parallel plate avalanche counters, and an ionization chamber with segmented anodes has been installed in the Broad Range Spectrometer at the Holifield Facility at Oak Ridge. The system, which has been designed for use with light-heavy ions with energies ranging from 10 to 25 MeV/amu, has a position resolution of approx. 0.1 mm, a scattering angle resolution of approx. 3 mrad, and a mass resolution of approx. 1/60.

  17. Method for detection and imaging over a broad spectral range

    DOEpatents

    Yefremenko, Volodymyr; Gordiyenko, Eduard; Pishko, legal representative, Olga; Novosad, Valentyn; Pishko, deceased; Vitalii

    2007-09-25

    A method of controlling the coordinate sensitivity in a superconducting microbolometer employs localized light, heating or magnetic field effects to form normal or mixed state regions on a superconducting film and to control the spatial location. Electron beam lithography and wet chemical etching were applied as pattern transfer processes in epitaxial Y--Ba--Cu--O films. Two different sensor designs were tested: (i) a 3 millimeter long and 40 micrometer wide stripe and (ii) a 1.25 millimeters long, and 50 micron wide meandering-like structure. Scanning the laser beam along the stripe leads to physical displacement of the sensitive area, and, therefore, may be used as a basis for imaging over a broad spectral range. Forming the superconducting film as a meandering structure provides the equivalent of a two-dimensional detector array. Advantages of this approach are simplicity of detector fabrication, and simplicity of the read-out process requiring only two electrical terminals.

  18. Microchip green laser sources: broad range of possibilities

    NASA Astrophysics Data System (ADS)

    Essaian, Stepan; Khaydarov, John; Slavov, Slav; Ter-Mikirtychev, Vartan; Gabrielyan, Gevorg; Keroopyan, Meruzhan; Soghomonyan, Suren

    2012-02-01

    Spectralus presents its progress in development of miniature, highly efficient, and versatile diode-pumped solid-state (DPSS) green laser source, based on a monolithic cavity microchip laser platform. The use of periodically poled MgO-doped Lithium Niobate (PPMgOLN) as the nonlinear frequency doubler together with gain material Nd3+:YVO4 allows obtaining a significant increase in the overall efficiency of the green microchip laser in comparison with other compact green laser source architectures with comparable output power. Originally, this laser source was designed to be part of the miniature and efficient RGB light source for microdisplay-based (LCOS, DLP or similar) mobile projector devices. Recently, we have extended range of operations for our original laser platform. In particular, we demonstrate the following: high peak power (>500mW), high average power (>200mW), broad temperature range of operation (-30°C - 60°C), and low noise CW operation (<0.5% RMS).

  19. Characterization and potential of three temperature ranges for hydrogen fermentation of cellulose by means of activity test and 16s rRNA sequence analysis.

    PubMed

    Gadow, Samir I; Jiang, Hongyu; Li, Yu-You

    2016-06-01

    A series of standardized activity experiments were performed to characterize three different temperature ranges of hydrogen fermentation from different carbon sources. 16S rRNA sequences analysis showed that the bacteria were close to Enterobacter genus in the mesophilic mixed culture (MMC) and Thermoanaerobacterium genus in the thermophilic and hyper-thermophilic mixed cultures (TMC and HMC). The MMC was able to utilize the glucose and cellulose to produce methane gas within a temperature range between 25 and 45 °C and hydrogen gas from 35 to 60°C. While, the TMC and HMC produced only hydrogen gas at all temperature ranges and the highest activity of 521.4mlH2/gVSSd was obtained by TMC. The thermodynamic analysis showed that more energy is consumed by hydrogen production from cellulose than from glucose. The experimental results could help to improve the economic feasibility of cellulosic biomass energy using three-phase technology to produce hythane.

  20. Microbiome characterization using SMRT sequencing on 16S rRNA genes across a range of amplicon sizes and variable region content

    USDA-ARS?s Scientific Manuscript database

    The sequence of variable regions along the 16S ribosomal RNA gene is often used to conduct metagenomic surveys of bacterial populations in specific habitats, because of the inter-species variability in these regions and because it is possible to design amplification primers in sections of the gene t...

  1. Broad tuning range filtering system with Optune interferometers

    NASA Astrophysics Data System (ADS)

    Miron, Nicolae

    2008-08-01

    An optical system built with two Optune interferometers cascaded according to Vernier principle has attractive tunable band pass filtering properties for numerous applications. Several characteristics of Optune interferometer such as 0.2 dB insertion loss flatness across at least 90 nm interval, no tuning holes across 240 nm tuning range, quasi-periodic free spectral range and 1 dB insertion loss are key parameters to obtain a cascade with 0.1 nm band pass tunable across minimum 90 nm. Several properties of Optune interferometers are analyzed to build a cascade tunable across minimum 90 nm: the relationships between the free spectral ranges, bandwidths and tuning conditions. It is presented also a cascade prototype with two interferometers having 9.72 nm free spectral range and respectively 11.12 nm free spectral range. The cascade band pass is 0.1 nm tunable with 1 pm accuracy to any arbitrary wavelength across 150 nm free spectral range, without any tuning hole. It has 0.125 ms / 100 nm tuning speed, the insertion loss is less than 3 dB, 50 dB contrast, 0.5 dB flatness and 0.2 dB polarization dependent loss. A controller based on digital signal processor monitors the operation of the cascade to achieve optimum tuning performance.

  2. Rainbow Vectors for Broad-Range Bacterial Fluorescence Labeling.

    PubMed

    Barbier, Mariette; Damron, F Heath

    2016-01-01

    Since their discovery, fluorescent proteins have been widely used to study protein function, localization or interaction, promoter activity and regulation, drug discovery or for non-invasive imaging. They have been extensively modified to improve brightness, stability, and oligomerization state. However, only a few studies have focused on understanding the dynamics of fluorescent proteins expression in bacteria. In this work, we developed a set plasmids encoding 12 fluorescent proteins for bacterial labeling to facilitate the study of pathogen-host interactions. These broad-spectrum plasmids can be used with a wide variety of Gram-negative microorganisms including Escherichia coli, Pseudomonas aeruginosa, Burkholderia cepacia, Bordetella bronchiseptica, Shigella flexneri or Klebsiella pneumoniae. For comparison, fluorescent protein expression and physical characteristics in Escherichia coli were analyzed using fluorescence microscopy, flow cytometry and in vivo imaging. Fluorescent proteins derived from the Aequorea Victoria family showed high photobleaching, while proteins form the Discosoma sp. and the Fungia coccina family were more photostable for microscopy applications. Only E2-Crimson, mCherry and mKeima were successfully detected for in vivo applications. Overall, E2-Crimson was the fastest maturing protein tested in E. coli with the best overall performance in the study parameters. This study provides a unified comparison and comprehensive characterization of fluorescent protein photostability, maturation and toxicity, and offers general recommendations on the optimal fluorescent proteins for in vitro and in vivo applications.

  3. Rainbow Vectors for Broad-Range Bacterial Fluorescence Labeling

    PubMed Central

    Barbier, Mariette; Damron, F. Heath

    2016-01-01

    Since their discovery, fluorescent proteins have been widely used to study protein function, localization or interaction, promoter activity and regulation, drug discovery or for non-invasive imaging. They have been extensively modified to improve brightness, stability, and oligomerization state. However, only a few studies have focused on understanding the dynamics of fluorescent proteins expression in bacteria. In this work, we developed a set plasmids encoding 12 fluorescent proteins for bacterial labeling to facilitate the study of pathogen-host interactions. These broad-spectrum plasmids can be used with a wide variety of Gram-negative microorganisms including Escherichia coli, Pseudomonas aeruginosa, Burkholderia cepacia, Bordetella bronchiseptica, Shigella flexneri or Klebsiella pneumoniae. For comparison, fluorescent protein expression and physical characteristics in Escherichia coli were analyzed using fluorescence microscopy, flow cytometry and in vivo imaging. Fluorescent proteins derived from the Aequorea Victoria family showed high photobleaching, while proteins form the Discosoma sp. and the Fungia coccina family were more photostable for microscopy applications. Only E2-Crimson, mCherry and mKeima were successfully detected for in vivo applications. Overall, E2-Crimson was the fastest maturing protein tested in E. coli with the best overall performance in the study parameters. This study provides a unified comparison and comprehensive characterization of fluorescent protein photostability, maturation and toxicity, and offers general recommendations on the optimal fluorescent proteins for in vitro and in vivo applications. PMID:26937640

  4. Laser-guide-stars used for cophasing broad capture ranges

    NASA Astrophysics Data System (ADS)

    Martinez, P.; Janin-Potiron, P.

    2016-08-01

    Context. Segmented primary mirrors are indispensable to master the steady increase in spatial resolution. Phasing optics systems must reduce segment misalignments to guarantee the high optical quality required for astronomical science programs. Aims: Modern telescopes routinely use adaptive optics systems to compensate for the atmosphere and use laser-guide-stars to create artificial stars as bright references in the field of observation. Because multiple laser-guide-star adaptive optics are being implemented in all major observatories, we propose to use man-made stars not only for adaptive optics, but for phasing optics. Methods: We propose a method called the doublet-wavelength coherence technique (DWCT), exploiting the D lines of sodium in the mesosphere using laser guide-stars. The signal coherence properties are then used. Results: The DWCT capture range exceeds current abilities by a factor of 100. It represents a change in paradigm by improving the phasing optics capture range from micrometric to millimetric. It thereby potentially eliminates the need of a man-made mechanical pre-phasing step. Conclusions: Extremely large telescopes require hundreds of segments, several of which need to be substituted on a daily basis to be recoated. The DWCT relaxes mechanical integration requirements and speeds up integration and re-integration process.

  5. BRAHMS (Broad Range Hadron Magnetic Spectrometer) Figures and Data Archive

    DOE Data Explorer

    The BRAHMS experiment was designed to measure charged hadrons over a wide range of rapidity and transverse momentum to study the reaction mechanisms of the relativistic heavy ion reactions at the Relativistic Heavy Ion Collider at Brookhaven National Laboratory and the properties of the highly excited nuclear matter formed in these reactions. The experiment took its first data during the RHIC 2000 year run and completed data taking in June 2006. The BRAHMS archive makes publications available and also makes data and figures from those publications available as separate items. See also the complete list of publications, multimedia presentations, and related papers at http://www4.rcf.bnl.gov/brahms/WWW/publications.html

  6. Bacterial diversity of soil in the vicinity of Pindari glacier, Himalayan mountain ranges, India, using culturable bacteria and soil 16S rRNA gene clones.

    PubMed

    Shivaji, S; Pratibha, M S; Sailaja, B; Hara Kishore, K; Singh, Ashish K; Begum, Z; Anarasi, Uttam; Prabagaran, S R; Reddy, G S N; Srinivas, T N R

    2011-01-01

    Three 16S rRNA gene clone libraries (P1L, P4L and P8L) were constructed using three soil samples (P1S, P4S and P8S) collected near Pindari glacier, Himalayas. The three libraries yielded a total of 703 clones. Actinobacteria, Firmicutes and Proteobacteria were common to the three libraries. In addition to the above P1L and P8L shared the phyla Acidobacteria, Bacteroidetes, Gemmatimonadetes and Planctomycetes. Phyla Chlamydiae, Chlorobi, Chloroflexi, Dictyoglomi, Fibrobacteres, Nitrospirae, Verrucomicrobia, candidate division SPAM and candidate TM7s TM7a phylum were present only in P1L. Rarefaction analysis indicated that the bacterial diversity in P4S and P8S soil samples was representative of the sample. Principal component analysis (PCA) revealed that P1S and P8S were different from P4S soil sample. PCA also indicated that arsenic content, pH, Cr and altitude influence the observed differences in the percentage of specific OTUs in the three 16S rRNA gene clone libraries. The observed bacterial diversity was similar to that observed for other Himalayan and non-polar cold habitats. A total of 40 strains of bacteria were isolated from the above three soil samples and based on the morphology 20 bacterial strains were selected for further characterization. The 20 bacteria belonged to 12 different genera. All the isolates were psychro-, halo- and alkalitolerant. Amylase and urease activities were detected in majority of the strains but lipase and protease activities were not detected. Long chain, saturated, unsaturated and branched fatty acids were predominant in the psychrotolerant bacteria.

  7. An efficient strategy for broad-range detection of low abundance bacteria without DNA decontamination of PCR reagents.

    PubMed

    Chang, Shy-Shin; Hsu, Hsung-Ling; Cheng, Ju-Chien; Tseng, Ching-Ping

    2011-01-01

    Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found. We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3'-end complementary to the template bacterial sequence and a 5'-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10-100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species. Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology

  8. An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

    PubMed Central

    Chang, Shy-Shin; Hsu, Hsung-Ling; Cheng, Ju-Chien; Tseng, Ching-Ping

    2011-01-01

    Background Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found. Methodology/Principal Findings We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3′-end complementary to the template bacterial sequence and a 5′-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10–100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species. Conclusions/Significance Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA

  9. Prevalence of Broad-Host-Range Lytic Bacteriophages of Sphaerotilus natans, Escherichia coli, and Pseudomonas aeruginosa

    PubMed Central

    Jensen, Ellen C.; Schrader, Holly S.; Rieland, Brenda; Thompson, Thomas L.; Lee, Kit W.; Nickerson, Kenneth W.; Kokjohn, Tyler A.

    1998-01-01

    Two bacteriophage collections were examined with regard to their ability to form plaques on multiple bacterial host species. Nine of 10 phages studied were found to be broad-host-range bacteriophages. These phages fell into two groups. Group 1, the SN series, was isolated from sewage treatment plant samples with Sphaerotilus natans ATCC 13338 as a host. The DNAs of these bacteriophages contained modified bases and were insensitive to cleavage by type I and II restriction endonucleases. The efficiency of plating of these bacteriophages was changed only slightly on the alternate host. Group 2, the BHR series, was isolated by a two-host enrichment protocol. These bacteriophages were sensitive to restriction, and their efficiency of plating was dramatically reduced on the alternate host. Our results suggest that a multiple-host enrichment protocol may be more effective for the isolation of broad-host-range bacteriophages by avoiding the selection bias inherent in single-host methods. At least two of the broad-host-range bacteriophages mediated generalized transduction. We suggest that broad-host-range bacteriophages play a key role in phage ecology and gene transfer in nature. PMID:9464396

  10. Complete genome sequence of the broad-host-range strain Sinorhizobium fredii USDA257

    USDA-ARS?s Scientific Manuscript database

    Here we announce the complete genome sequence of the symbiotic and nitrogen fixing bacterium Sinorhizobium fredii USDA257. The genome shares a high degree of similarity with the closely related broad-host-range strains S. fredii NGR234 and HH103. Most striking, the USDA257 genome encodes for a wealt...

  11. Draft genome sequence of Pseudomonas fuscovaginae, a broad-host-range pathogen of plants.

    PubMed

    Patel, Hitendra Kumar; da Silva, Daniel Passos; Devescovi, Giulia; Maraite, Henri; Paszkiewicz, Konrad; Studholme, David J; Venturi, Vittorio

    2012-05-01

    Pseudomonas fuscovaginae was first reported as a pathogen of rice causing sheath rot in plants grown at high altitudes. P. fuscovaginae is now considered a broad-host-range plant pathogen causing disease in several economically important plants. We report what is, to our knowledge, the first draft genome sequence of a P. fuscovaginae strain.

  12. Draft Genome Sequence of Pseudomonas fuscovaginae, a Broad-Host-Range Pathogen of Plants

    PubMed Central

    Patel, Hitendra Kumar; Passos da Silva, Daniel; Devescovi, Giulia; Maraite, Henri; Paszkiewicz, Konrad; Studholme, David J.

    2012-01-01

    Pseudomonas fuscovaginae was first reported as a pathogen of rice causing sheath rot in plants grown at high altitudes. P. fuscovaginae is now considered a broad-host-range plant pathogen causing disease in several economically important plants. We report what is, to our knowledge, the first draft genome sequence of a P. fuscovaginae strain. PMID:22535942

  13. Monolithic front-end preamplifiers for a broad range of calorimetry applications

    SciTech Connect

    Radeka, V.; Rescia, S.; Manfredi, P.F.; Speziali, V. |

    1993-12-31

    The present paper summarizes the salient results of a research and development activity in the area of low noise preamplifiers for different applications in calorimetry. Design target for all circuits considered here are low noise, ability to cope with broad energy ranges and radiation hardness.

  14. Narrow- and Broad-Host-Range Symbiotic Plasmids of Rhizobium spp. Strains That Nodulate Phaseolus vulgaris

    PubMed Central

    Brom, Susana; Martinez, Esperanza; Dávila, Guillermo; Palacios, Rafael

    1988-01-01

    Agrobacterium transconjugants containing symbiotic plasmids from different Rhizobium spp. strains that nodulate Phaseolus vulgaris were obtained. All transconjugants conserved the parental nodulation host range. Symbiotic (Sym) plasmids of Rhizobium strains isolated originally from P. vulgaris nodules, which had a broad nodulation host range, and single-copy nitrogenase genes conferred a Fix+ phenotype to the Agrobacterium transconjugants. A Fix− phenotype was obtained with Sym plasmids of strains isolated from P. vulgaris nodules that had a narrow host range and reiterated nif genes, as well as with Sym plasmids of strains isolated from other legumes that presented single nif genes and a broad nodulation host range. This indicates that different types of Sym plasmids can confer the ability to establish an effective symbiosis with P. vulgaris. Images PMID:16347637

  15. Prosthetic Joint Infection Diagnosis Using Broad-Range PCR of Biofilms Dislodged from Knee and Hip Arthroplasty Surfaces Using Sonication

    PubMed Central

    Gomez, Eric; Cazanave, Charles; Cunningham, Scott A.; Greenwood-Quaintance, Kerryl E.; Steckelberg, James M.; Uhl, James R.; Hanssen, Arlen D.; Karau, Melissa J.; Schmidt, Suzannah M.; Osmon, Douglas R.; Berbari, Elie F.; Mandrekar, Jayawant

    2012-01-01

    Periprosthetic tissue and/or synovial fluid PCR has been previously studied for prosthetic joint infection (PJI) diagnosis; however, few studies have assessed the utility of PCR on biofilms dislodged from the surface of explanted arthroplasties using vortexing and sonication (i.e., sonicate fluid PCR). We compared sonicate fluid 16S rRNA gene real-time PCR and sequencing to culture of synovial fluid, tissue, and sonicate fluid for the microbiologic diagnosis of PJI. PCR sequences generating mixed chromatograms were decatenated using RipSeq Mixed. We studied sonicate fluids from 135 and 231 subjects with PJI and aseptic failure, respectively. Synovial fluid, tissue, and sonicate fluid culture and sonicate fluid PCR had similar sensitivities (64.7, 70.4, 72.6, and 70.4%, respectively; P > 0.05) and specificities (96.9, 98.7, 98.3, and 97.8%, respectively; P > 0.05). Combining sonicate fluid culture and PCR, the sensitivity was higher (78.5%, P < 0.05) than those of individual tests, with similar specificity (97.0%). Thirteen subjects had positive sonicate fluid culture but negative PCR, and 11 had negative sonicate fluid culture but positive PCR (among which 7 had prior use of antimicrobials). Broad-range PCR and culture of sonicate fluid have equivalent performance for PJI diagnosis. PMID:22895042

  16. Dielectric relaxation of relaxor ferroelectric P(VDF-TrFE-CFE) terpolymer over broad frequency range.

    PubMed

    Wang, Yong; Lu, Sheng-Guo; Lanagan, Michael; Zhang, Qiming

    2009-03-01

    Dielectric properties of a relaxor ferroelectric polymer, poly(vinylidene fluoride-trifluoroethylene-chlorofluoroethylene) [P(VDF-TrFE-CFE)] terpolymer, were investigated over a broad range of frequency (from 0.1 kHz to 1 GHz) and a broad range of temperature (-20 degrees C to 76 degrees C). Time-temperature superposition was used to extrapolate the dielectric constant to high frequencies (approximately 1 GHz) from low frequency data (1 MHz). The consistency between the directly measured and the extrapolated data indicate that the time-temperature superposition can be applied at temperature ranging from the glass transition to the broad ferroelectric-paraelectric transition peak of relaxor, indicating that the glass transition is still the dominating relaxation process at room temperature for the ferroelectric relaxor. Compared with the dielectric properties of poly(vinylidene fluoride-trifluoroethylene) [P(VDF-TrFE] copolymer, the terpolymer shows a higher dielectric constant even at 1 GHz, which is considered to originate from the random defects modification converting the long-chain polar-molecular conformation to short-range molecular microstructures and enhancing the molecular motions in both polar and nonpolar nanodomains.

  17. Collection of small subunit (16S- and 16S-like) ribosomal RNA structures: 1994.

    PubMed Central

    Gutell, R R

    1994-01-01

    A collection of diverse 16S and 16S-like rRNA secondary structure diagrams are available. This set of rRNAs contains representative structures from all of the major phylogenetic groupings--Archaea, (eu)Bacteria, and the nucleus, mitochondrion, and chloroplast of Eucarya. Within this broad phylogenetic sampling are examples of the major forms of structural diversity currently known for this class of rRNAs. These structure diagrams are available online through our computer-network WWW server and anonymous ftp, as well as from the author in hardcopy format. PMID:7524024

  18. 2-Arylindoles: a privileged molecular scaffold with potent, broad-ranging pharmacological activity.

    PubMed

    Lal, S; Snape, T J

    2012-01-01

    Privileged structures bind to multiple receptors with high affinity, thus aiding the development of novel biologically active compounds. Indoles are classed as privileged structures, and as a result of the indole nucleus being present in a broad range of biologically active molecules, it has been suggested that indoles probably represent the most important of all structural classes in drug discovery. Amongst the indole class of compounds is a particular subset - 2-arylindoles - which appear to be a most promising lead for drug development. This review summarises the wide-ranging activities of 2-arylindoles and some of their important biological activities reported in the literature over the past two decades.

  19. All-fiber broad-range self-sweeping Yb-doped fiber laser

    NASA Astrophysics Data System (ADS)

    Lobach, Ivan A.; Kablukov, Sergey A.; Podivilov, Evgeniy V.; Babin, Sergey A.

    2012-02-01

    The effect of broad-range self-sweeping in Yb-doped fiber laser has been demonstrated experimentally for the first time. The self-sweeping effect is observed in an all-fiber laser configuration with a double-clad Yb-doped fiber and a cavity formed by a broad-band fiber loop mirror and Fresnel reflection from one cleaved end. The sweep range is limited by the width of the broad-band reflector and reaches up to 16nm. It is found that the self-sweeping effect is related to selfpulsations. So the sweep rate is increased with an increase in pump power and is decreased with increasing cavity length. RF and optical spectra (linewidth is measured to be not more than 100 MHz) show that during the evolution of a single pulse a small number of longitudinal modes take a part in lasing. Based on these results we propose a model describing dynamics of the laser frequency. The model is based on the spatial hole burning effect and the gain saturation in Yb laser transition, and takes into account self-pulsations of the laser. Theoretical estimation for pulse to pulse change of lasing frequency is in good agreement with experimental data.

  20. Bacteriophages isolated from Lake Michigan demonstrate broad host-range across several bacterial phyla.

    PubMed

    Malki, Kema; Kula, Alex; Bruder, Katherine; Sible, Emily; Hatzopoulos, Thomas; Steidel, Stephanie; Watkins, Siobhan C; Putonti, Catherine

    2015-10-09

    The study of bacteriophages continues to generate key information about microbial interactions in the environment. Many phenotypic characteristics of bacteriophages cannot be examined by sequencing alone, further highlighting the necessity for isolation and examination of phages from environmental samples. While much of our current knowledge base has been generated by the study of marine phages, freshwater viruses are understudied in comparison. Our group has previously conducted metagenomics-based studies samples collected from Lake Michigan - the data presented in this study relate to four phages that were extracted from the same samples. Four phages were extracted from Lake Michigan on the same bacterial host, exhibiting similar morphological characteristics as shown under transmission electron microscopy. Growth characteristics of the phages were unique to each isolate. Each phage demonstrated a host-range spanning several phyla of bacteria - to date, such a broad host-range is yet to be reported. Genomic data reveals genomes of a similar size, and close similarities between the Lake Michigan phages and the Pseudomonas phage PB1, however, the majority of annotated genes present were ORFans and little insight was offered into mechanisms for host-range. The phages isolated from Lake Michigan are capable of infecting several bacterial phyla, and demonstrate varied phenotypic characteristics despite similarities in host preference, and at the genomic level. We propose that such a broad host-range is likely related to the oligotrophic nature of Lake Michigan, and the competitive benefit that this characteristic may lend to phages in nature.

  1. Broad-host-range IncP-1 plasmids and their resistance potential

    PubMed Central

    Popowska, Magdalena; Krawczyk-Balska, Agata

    2013-01-01

    The plasmids of the incompatibility (Inc) group IncP-1, also called IncP, as extrachromosomal genetic elements can transfer and replicate virtually in all Gram-negative bacteria. They are composed of backbone genes that encode a variety of essential functions and accessory genes that have implications for human health and environmental bioremediation. Broad-host-range IncP plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to a broad spectrum of antibiotics, heavy metals, and quaternary ammonium compounds used as disinfectants. The backbone of these plasmids carries modules that enable them to effectively replicate, move to a new host via conjugative transfer and to be stably maintained in bacterial cells. The adaptive, resistance, and virulence genes are mainly located on mobile genetic elements integrated between the functional plasmid backbone modules. Environmental studies have demonstrated the wide distribution of IncP-like replicons in manure, soils and wastewater treatment plants. They also are present in strains of pathogenic or opportunistic bacteria, which can be a cause for concern, because they may encode multiresistance. Their broad distribution suggests that IncP plasmids play a crucial role in bacterial adaptation by utilizing horizontal gene transfer. This review summarizes the variety of genetic information and physiological functions carried by IncP plasmids, which can contribute to the spread of antibiotic and heavy metal resistance while also mediating the process of bioremediation of pollutants. Due to the location of the resistance genes on plasmids with a broad-host-range and the presence of transposons carrying these genes it seems that the spread of these genes would be possible and quite hazardous in infection control. Future studies are required to determine the level of risk of the spread of resistance genes located on these plasmids. PMID:23471189

  2. Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

    PubMed Central

    Klümper, Uli; Riber, Leise; Dechesne, Arnaud; Sannazzarro, Analia; Hansen, Lars H; Sørensen, Søren J; Smets, Barth F

    2015-01-01

    Conjugal plasmids can provide microbes with full complements of new genes and constitute potent vehicles for horizontal gene transfer. Conjugal plasmid transfer is deemed responsible for the rapid spread of antibiotic resistance among microbes. While broad host range plasmids are known to transfer to diverse hosts in pure culture, the extent of their ability to transfer in the complex bacterial communities present in most habitats has not been comprehensively studied. Here, we isolated and characterized transconjugants with a degree of sensitivity not previously realized to investigate the transfer range of IncP- and IncPromA-type broad host range plasmids from three proteobacterial donors to a soil bacterial community. We identified transfer to many different recipients belonging to 11 different bacterial phyla. The prevalence of transconjugants belonging to diverse Gram-positive Firmicutes and Actinobacteria suggests that inter-Gram plasmid transfer of IncP-1 and IncPromA-type plasmids is a frequent phenomenon. While the plasmid receiving fractions of the community were both plasmid- and donor- dependent, we identified a core super-permissive fraction that could take up different plasmids from diverse donor strains. This fraction, comprising 80% of the identified transconjugants, thus has the potential to dominate IncP- and IncPromA-type plasmid transfer in soil. Our results demonstrate that these broad host range plasmids have a hitherto unrecognized potential to transfer readily to very diverse bacteria and can, therefore, directly connect large proportions of the soil bacterial gene pool. This finding reinforces the evolutionary and medical significances of these plasmids. PMID:25333461

  3. [Development of the biological preparation enatin with broad-range antimicrobial action].

    PubMed

    Romanovskaia, T V; Kolomiets, E I; Zdor, N A; Lobanok, A G

    2002-01-01

    Physiological and biochemical traits of epiphytic spore forming bacteria Bacillus pumilis BIM V-263 were examined. The nutrient medium and conditions for submerged cultivation of the strain were selected. The growth dynamics and antagonistic activity during cultivation in a laboratory fermenter ANKUM-2M were studied. The results provide grounds for development of the biological preparation Enatin with broad-range antimicrobial effect. The plant-protective and growth-stimulating effect of Enatin was examined in laboratory and field experiments. The preparation holds promise as means for biological control of crop pathogens.

  4. Elastic 'tethers' connect separating anaphase chromosomes in a broad range of animal cells.

    PubMed

    Forer, Arthur; Duquette, Michelle L; Paliulis, Leocadia V; Fegaras, E; Ono, M; Preece, D; Berns, Michael W

    2017-09-01

    We describe the general occurrence in animal cells of elastic components ("tethers") that connect individual chromosomes moving to opposite poles during anaphase. Tethers, originally described in crane-fly spermatocytes, exert force on chromosome arms opposite to the direction the anaphase chromosomes move. We show that they exist in a broad range of animal cells. Thus tethers are previously unrecognised components of general mitotic mechanisms that exert force on chromosomes and they need to be accounted for in general models of mitosis in terms of forces on chromosomes and in terms of what their roles might be. Copyright © 2017 Elsevier GmbH. All rights reserved.

  5. Characterisation of Host Growth after Infection with a Broad-Range Freshwater Cyanopodophage

    PubMed Central

    Watkins, Siobhan C.; Smith, James R.; Hayes, Paul K.; Watts, Joy E. M.

    2014-01-01

    Freshwater cyanophages are poorly characterised in comparison to their marine counterparts, however, the level of genetic diversity that exists in freshwater cyanophage communities is likely to exceed that found in marine environments, due to the habitat heterogeneity within freshwater systems. Many cyanophages are specialists, infecting a single host species or strain; however, some are less fastidious and infect a number of different host genotypes within the same species or even hosts from different genera. Few instances of host growth characterisation after infection by broad host-range phages have been described. Here we provide an initial characterisation of interactions between a cyanophage isolated from a freshwater fishing lake in the south of England and its hosts. Designated ΦMHI42, the phage is able to infect isolates from two genera of freshwater cyanobacteria, Planktothrix and Microcystis. Transmission Electron Microscopy and Atomic Force Microscopy indicate that ΦMHI42 is a member of the Podoviridae, albeit with a larger than expected capsid. The kinetics of host growth after infection with ΦMHI42 differed across host genera, species and strains in a way that was not related to the growth rate of the uninfected host. To our knowledge, this is the first characterisation of the growth of cyanobacteria in the presence of a broad host-range freshwater cyanophage. PMID:24489900

  6. Identification of virulence associated loci in the emerging broad host range plant pathogen Pseudomonas fuscovaginae.

    PubMed

    Patel, Hitendra Kumar; Matiuzzo, Maura; Bertani, Iris; Bigirimana, Vincent de Paul; Ash, Gavin J; Höfte, Monica; Venturi, Vittorio

    2014-11-14

    Pseudomonas fuscovaginae (Pfv) is an emerging plant pathogen of rice and also of other gramineae plants. It causes sheath brown rot disease in rice with symptoms that are characterized by brown lesions on the flag leaf sheath, grain discoloration and sterility. It was first isolated as a high altitude pathogen in Japan and has since been reported in several countries throughout the world. Pfv is a broad host range pathogen and very little is known about its virulence mechanisms. An in planta screen of 1000 random independent Tn5 genomic mutants resulted in the isolation of nine mutants which showed altered virulence. Some of these isolates are mutated for functions which are known to be virulence associated factors in other phytopathogenic bacteria (eg. pil gene, phytotoxins and T6SS) and others might represent novel virulence loci. Being an emerging pathogen worldwide, the broad host range pathogen Pfv has not yet been studied for its virulence functions. The roles of the nine loci identified in the in planta screen are discussed in relation to pathogenicity of Pfv. In summary, this article reports a first study on the virulence of this pathogen involving in planta screening studies and suggests the presence of several virulence features with known and novel functions in the Pseudomonas group of bacteria.

  7. Convenient broad-host-range unstable vectors for studying stabilization cassettes in diverse bacteria.

    PubMed

    Bartosik, Aneta A; Glabski, Krzysztof; Kulinska, Anna; Lewicka, Ewa; Godziszewska, Jolanta; Markowska, Aleksandra; Jagura-Burdzy, Grazyna

    2016-04-05

    Low-copy-number vectors of potential wide application in biotechnology need to encode stabilization modules ensuring their stable inheritance. The efficiency of stabilization may vary depending on the plasmid host so a thorough analysis of stabilization functions is required before use. To facilitate such analysis highly unstable, mobilizable, broad-host-range (BHR) vectors based on RK2 replicon were constructed. The vectors are suitable for testing of various stabilization functions, including plasmid and chromosomal partitioning cassettes encoding ParB homologues capable of spreading on DNA. The xylE or lacZ reporter systems facilitate easy monitoring of plasmid segregation. The range of BHR vectors with different reporter cassettes and alternative mobilization systems expands their application in diverse bacterial species.

  8. Nonlinear effects in photoionization over a broad photon-energy range within the TDCIS scheme

    NASA Astrophysics Data System (ADS)

    Karamatskou, Antonia

    2017-01-01

    The present tutorial provides an overview of the time-dependent configuration interaction singles scheme applied to nonlinear ionization over a broad photon-energy range. The efficient propagation of the wave function and the calculation of photoelectron spectra within this approach are described and demonstrated in various applications. Above-threshold ionization of argon and xenon in the extreme ultraviolet energy range is investigated as an example. A particular focus is put on the xenon 4d giant dipole resonance and the information that nonlinear ionization can provide about resonance substructure. Furthermore, above-threshold ionization is studied in the x-ray regime and the intensity regime, at which multiphoton ionization starts to play a role at hard x-ray photon energies, is identified.

  9. Time-of-flight electron spectrometer for a broad range of kinetic energies

    SciTech Connect

    Kothe, Alexander; Metje, Jan; Wilke, Martin; Moguilevski, Alexandre; Engel, Nicholas; Al-Obaidi, Ruba; Richter, Clemens; Golnak, Ronny; Kiyan, Igor Yu.; Aziz, Emad F.

    2013-02-15

    A newly constructed time-of-flight electron spectrometer of the magnetic bottle type is characterized for electron detection in a broad range of kinetic energies. The instrument is designed to measure the energy spectra of electrons generated from liquids excited by strong laser fields and photons in the range of extreme ultra violet and soft X-rays. Argon inner shell electrons were recorded to calibrate the spectrometer and investigate its characteristics, such as energy resolution and collection efficiency. Its energy resolution {Delta}E/E of 1.6% allows resolving the Ar 2p spin orbit structure at kinetic energies higher than 100 eV. The collection efficiency is determined and compared to that of the spectrometer in its field-free configuration.

  10. Time-of-flight electron spectrometer for a broad range of kinetic energies.

    PubMed

    Kothe, Alexander; Metje, Jan; Wilke, Martin; Moguilevski, Alexandre; Engel, Nicholas; Al-Obaidi, Ruba; Richter, Clemens; Golnak, Ronny; Kiyan, Igor Yu; Aziz, Emad F

    2013-02-01

    A newly constructed time-of-flight electron spectrometer of the magnetic bottle type is characterized for electron detection in a broad range of kinetic energies. The instrument is designed to measure the energy spectra of electrons generated from liquids excited by strong laser fields and photons in the range of extreme ultra violet and soft X-rays. Argon inner shell electrons were recorded to calibrate the spectrometer and investigate its characteristics, such as energy resolution and collection efficiency. Its energy resolution ΔE/E of 1.6% allows resolving the Ar 2p spin orbit structure at kinetic energies higher than 100 eV. The collection efficiency is determined and compared to that of the spectrometer in its field-free configuration.

  11. Genetic interactions of broad host-range plasmid RK2: evidence for a complex replication regulon.

    PubMed

    Figurski, D H; Young, C; Schreiner, H C; Pohlman, R F; Bechhofer, D H; Prince, A S; D'Amico, T F

    1985-01-01

    The kil and kor genes of RK2 are novel genetic determinants further that the kil and kor network constitutes a replication regulon, and that perhaps the function of this regulon is to ensure expression of trfA at appropriate levels. The complexity of this regulon may reflect an ability of the system to adapt to the intracellular environments of a variety of hosts. Indeed, there is tantalizing evidence that regions encoding kil or kor genes are important to host range (1,2,6,28; Schmidhauser and Helinski, pers. comm.). We are therefore hopeful that the study of these genes and the eventual determination of the molecular basis of their actions will lead to a complete understanding of the replication control and broad host range capability of IncP plasmids.

  12. Broad-range lytic bacteriophages that kill Staphylococcus aureus local field strains.

    PubMed

    Abatángelo, Virginia; Peressutti Bacci, Natalia; Boncompain, Carina A; Amadio, Ariel A; Carrasco, Soledad; Suárez, Cristian A; Morbidoni, Héctor R

    2017-01-01

    Staphylococcus aureus is a very successful opportunistic pathogen capable of causing a variety of diseases ranging from mild skin infections to life-threatening sepsis, meningitis and pneumonia. Its ability to display numerous virulence mechanisms matches its skill to display resistance to several antibiotics, including β-lactams, underscoring the fact that new anti-S. aureus drugs are urgently required. In this scenario, the utilization of lytic bacteriophages that kill bacteria in a genus -or even species- specific way, has become an attractive field of study. In this report, we describe the isolation, characterization and sequencing of phages capable of killing S. aureus including methicillin resistant (MRSA) and multi-drug resistant S. aureus local strains from environmental, animal and human origin. Genome sequencing and bio-informatics analysis showed the absence of genes encoding virulence factors, toxins or antibiotic resistance determinants. Of note, there was a high similarity between our set of phages to others described in the literature such as phage K. Considering that reported phages were obtained in different continents, it seems plausible that there is a commonality of genetic features that are needed for optimum, broad host range anti-staphylococcal activity of these related phages. Importantly, the high activity and broad host range of one of our phages underscores its promising value to control the presence of S. aureus in fomites, industry and hospital environments and eventually on animal and human skin. The development of a cocktail of the reported lytic phages active against S. aureus-currently under way- is thus, a sensible strategy against this pathogen.

  13. Broad-range lytic bacteriophages that kill Staphylococcus aureus local field strains

    PubMed Central

    Boncompain, Carina A.; Amadio, Ariel A.; Carrasco, Soledad; Suárez, Cristian A.

    2017-01-01

    Staphylococcus aureus is a very successful opportunistic pathogen capable of causing a variety of diseases ranging from mild skin infections to life-threatening sepsis, meningitis and pneumonia. Its ability to display numerous virulence mechanisms matches its skill to display resistance to several antibiotics, including β-lactams, underscoring the fact that new anti-S. aureus drugs are urgently required. In this scenario, the utilization of lytic bacteriophages that kill bacteria in a genus -or even species- specific way, has become an attractive field of study. In this report, we describe the isolation, characterization and sequencing of phages capable of killing S. aureus including methicillin resistant (MRSA) and multi-drug resistant S. aureus local strains from environmental, animal and human origin. Genome sequencing and bio-informatics analysis showed the absence of genes encoding virulence factors, toxins or antibiotic resistance determinants. Of note, there was a high similarity between our set of phages to others described in the literature such as phage K. Considering that reported phages were obtained in different continents, it seems plausible that there is a commonality of genetic features that are needed for optimum, broad host range anti-staphylococcal activity of these related phages. Importantly, the high activity and broad host range of one of our phages underscores its promising value to control the presence of S. aureus in fomites, industry and hospital environments and eventually on animal and human skin. The development of a cocktail of the reported lytic phages active against S. aureus–currently under way- is thus, a sensible strategy against this pathogen. PMID:28742812

  14. Bioorthogonally cross-linked hydrogel network with precisely controlled disintegration time over a broad range.

    PubMed

    Xu, Jianwen; Feng, Ellva; Song, Jie

    2014-03-19

    Hydrogels with predictable degradation are highly desired for biomedical applications where timely disintegration of the hydrogel (e.g., drug delivery, guided tissue regeneration) is required. However, precisely controlling hydrogel degradation over a broad range in a predictable manner is challenging due to limited intrinsic variability in the degradation rate of liable bonds and difficulties in modeling degradation kinetics for complex polymer networks. More often than not, empirical tuning of the degradation profile results in undesired changes in other properties. Here we report a simple but versatile hydrogel platform that allows us to formulate hydrogels with predictable disintegration time from 2 to >250 days yet comparable macroscopic physical properties. This platform is based on a well-defined network formed by two pairs of four-armed polyethylene glycol macromers terminated with azide and dibenzocyclooctyl groups, respectively, via labile or stable linkages. The high-fidelity bioorthogonal reaction between the symmetric hydrophilic macromers enables robust cross-linking in water, phosphate-buffered saline, and cell culture medium to afford tough hydrogels capable of withstanding >90% compressive strain. Strategic placement of labile ester linkages near the cross-linking site within this superhydrophilic network, accomplished by adjustments of the ratio of the macromers used, enables broad tuning of the disintegration rates precisely matching with the theoretical predictions based on first-order linkage cleavage kinetics. This platform can be exploited for applications where a precise degradation rate is targeted.

  15. Bioorthogonally Cross-Linked Hydrogel Network with Precisely Controlled Disintegration Time over a Broad Range

    PubMed Central

    2015-01-01

    Hydrogels with predictable degradation are highly desired for biomedical applications where timely disintegration of the hydrogel (e.g., drug delivery, guided tissue regeneration) is required. However, precisely controlling hydrogel degradation over a broad range in a predictable manner is challenging due to limited intrinsic variability in the degradation rate of liable bonds and difficulties in modeling degradation kinetics for complex polymer networks. More often than not, empirical tuning of the degradation profile results in undesired changes in other properties. Here we report a simple but versatile hydrogel platform that allows us to formulate hydrogels with predictable disintegration time from 2 to >250 days yet comparable macroscopic physical properties. This platform is based on a well-defined network formed by two pairs of four-armed polyethylene glycol macromers terminated with azide and dibenzocyclooctyl groups, respectively, via labile or stable linkages. The high-fidelity bioorthogonal reaction between the symmetric hydrophilic macromers enables robust cross-linking in water, phosphate-buffered saline, and cell culture medium to afford tough hydrogels capable of withstanding >90% compressive strain. Strategic placement of labile ester linkages near the cross-linking site within this superhydrophilic network, accomplished by adjustments of the ratio of the macromers used, enables broad tuning of the disintegration rates precisely matching with the theoretical predictions based on first-order linkage cleavage kinetics. This platform can be exploited for applications where a precise degradation rate is targeted. PMID:24597638

  16. Broad-host-range plasmid vectors for gene expression in bacteria.

    PubMed

    Lale, Rahmi; Brautaset, Trygve; Valla, Svein

    2011-01-01

    This chapter provides methods and insights into the use of broad-host-range plasmid vectors useful for expression of genes in a variety of bacteria. The main focus is on IncQ, IncW, IncP, and pBBR1-based plasmids which have all been used for such applications. The specific design of each vector is adapted to its use, and here we describe, as an example, a protocol for construction (in Escherichia coli) of large insert DNA libraries in an IncP type vector and transfer of the library to the desired host. The genes of interest will in this case have to be expressed from their own promoters and the libraries will be screened by a method that best fits the functions of the gene or gene clusters searched for.

  17. Nanoporous Anodic Alumina 3D FDTD Modelling for a Broad Range of Inter-pore Distances

    NASA Astrophysics Data System (ADS)

    Bertó-Roselló, Francesc; Xifré-Pérez, Elisabet; Ferré-Borrull, Josep; Pallarès, Josep; Marsal, Lluis F.

    2016-08-01

    The capability of the finite difference time domain (FDTD) method for the numerical modelling of the optical properties of nanoporous anodic alumina (NAA) in a broad range of inter-pore distances is evaluated. FDTD permits taking into account in the same numerical framework all the structural features of NAA, such as the texturization of the interfaces or the incorporation of electrolyte anions in the aluminium oxide host. The evaluation is carried out by comparing reflectance measurements from two samples with two very different inter-pore distances with the simulation results. Results show that considering the texturization is crucial to obtain good agreement with the measurements. On the other hand, including the anionic layer in the model leads to a second-order contribution to the reflectance spectrum.

  18. Sequences of versatile, broad-host-range vectors of the RK2 family.

    SciTech Connect

    Scott, H. N.; Laible, P. D.; Hanson, D. K.; Biosciences Division

    2003-07-01

    Plasmid pRK404-a smaller derivative of RK2-is a tetracycline-resistant broad-host-range vector that carries a multiple cloning site and the lacZ(alpha) peptide that enables blue/white selection for cloned inserts in Escherichia coli. We present herein the complete and annotated sequence of pRK404 and three related vectors-pRK437, pRK442, and pRK442(H). These derivatives have proven to be valuable tools for genetic manipulation in Gram-negative bacteria. The knowledge of their complete sequences will facilitate efficient future engineering of them and will enhance their general applicability to the design of genetic systems for use in organisms for which new genomic sequence data are becoming available.

  19. Evolution of broad host range in retroviruses leads to cell death mediated by highly cytopathic variants.

    PubMed

    Rainey, G Jonah A; Coffin, John M

    2006-01-01

    The ability of many retroviruses to cause disease can be correlated to their cytopathic effect (CPE) in tissue culture characterized by an acute period of cell death and viral DNA accumulation. Here, we show that mutants of a subgroup B avian retrovirus (Alpharetrovirus) cause a very dramatic CPE in certain susceptible avian cells that is coincident with elevated levels of apoptosis, as measured by nuclear morphology, and persistent viral DNA accumulation. These mutants also have a broadly extended host range that includes rodent, cat, dog, monkey, and human cells (31). Previously, we have shown that the mutants exhibit diminished resistance to superinfection. The results presented here have important implications for the process of evolution of retroviruses to use distinct cellular receptors.

  20. Peracetic Acid Treatment Generates Potent Inactivated Oral Vaccines from a Broad Range of Culturable Bacterial Species

    PubMed Central

    Moor, Kathrin; Wotzka, Sandra Y.; Toska, Albulena; Diard, Médéric; Hapfelmeier, Siegfried; Slack, Emma

    2016-01-01

    Our mucosal surfaces are the main sites of non-vector-borne pathogen entry, as well as the main interface with our commensal microbiota. We are still only beginning to understand how mucosal adaptive immunity interacts with commensal and pathogenic microbes to influence factors such as infectivity, phenotypic diversity, and within-host evolution. This is in part due to difficulties in generating specific mucosal adaptive immune responses without disrupting the mucosal microbial ecosystem itself. Here, we present a very simple tool to generate inactivated mucosal vaccines from a broad range of culturable bacteria. Oral gavage of 1010 peracetic acid-inactivated bacteria induces high-titer-specific intestinal IgA in the absence of any measurable inflammation or species invasion. As a proof of principle, we demonstrate that this technique is sufficient to provide fully protective immunity in the murine model of invasive non-typhoidal Salmonellosis, even in the face of severe innate immune deficiency. PMID:26904024

  1. Nanoporous Anodic Alumina 3D FDTD Modelling for a Broad Range of Inter-pore Distances.

    PubMed

    Bertó-Roselló, Francesc; Xifré-Pérez, Elisabet; Ferré-Borrull, Josep; Pallarès, Josep; Marsal, Lluis F

    2016-12-01

    The capability of the finite difference time domain (FDTD) method for the numerical modelling of the optical properties of nanoporous anodic alumina (NAA) in a broad range of inter-pore distances is evaluated. FDTD permits taking into account in the same numerical framework all the structural features of NAA, such as the texturization of the interfaces or the incorporation of electrolyte anions in the aluminium oxide host. The evaluation is carried out by comparing reflectance measurements from two samples with two very different inter-pore distances with the simulation results. Results show that considering the texturization is crucial to obtain good agreement with the measurements. On the other hand, including the anionic layer in the model leads to a second-order contribution to the reflectance spectrum.

  2. Jet-Cooled Broad Range Near-Ir Scan of Reactive Intermediates Using Cavity Ringdown Spectroscopy

    NASA Astrophysics Data System (ADS)

    Kline, Neal D.; Codd, Terrance J.; Chen, Ming-Wei; Miller, Terry A.

    2012-06-01

    A technique has been developed for obtaining broad range scans of jet-cooled radicals using cavity ringdown spectroscopy. The talk will describe a method that uses a slit jet expansion in tandem with an electrical discharge to produce the reactive intermediates and obtain rotational temperatures of 15-30 K and effective vibrational temperatures of 0 K. The spectrum can be recorded by scanning the second stokes of an H_2 Raman shifted YAG-pumped dye laser with bandwidth of ≈ 0.1 cm-1. This technique has been used to obtain the jet-cooled widetilde{A} - widetilde{X} spectra of the NO_3 radical and the 2,1-hydroxypropyl peroxy radical (2,1-HPP). Obtaining the jet-cooled spectra helped to identify hot bands present in the room temperature spectrum of NO_3 and also helped to identify cold, low-frequency fundamental bands present in the 2,1-HPP spectrum.

  3. Lateral mixing in the eddying regime and a new broad-ranging formulation

    NASA Astrophysics Data System (ADS)

    Hecht, Matthew W.; Petersen, Mark R.; Wingate, Beth A.; Hunke, Elizabeth; Maltrud, Mathew

    We survey a number of issues associated with lateral dissipation in eddy-resolving ocean models and present two effective techniques. The first is a specification of lateral viscosity that is closely related to that of Chassignet and Garraffo [2001] involving the combined application of biharmonic and Laplacian forms of viscosity. The specification can in principle be applied across a broad range of model resolution, although our testing was performed only at eddy-resolving scale where a relatively simple form suffices. The second is the implementation of the Lagrangian averaged Navier-Stokes alpha (LANS-α) subgridscale turbulence scheme in a primitive equation ocean model, with our presentation here being largely a summary of the recent work of Hecht et al., [2008] and Petersen et al. [2008]. As an inherently non-dissipative turbulence parameterization, one can understand the higher levels of eddy variability with LANS-α as coming about through an increase in the effective Rossby radius of deformation

  4. Monoglyceride-based organogelator for broad-range oil uptake with high capacity.

    PubMed

    Wang, Dong; Niu, Jian; Wang, Zhenggong; Jin, Jian

    2015-02-10

    Oil/water separation has been a worldwide subject because of increasing release of oil-containing wastewater as well as several marine oil spills. The phase-selective organogelators (PSOGs) are thought to offer a potential and effective implement for addressing this issue. An ideal PSOG for oil adsorption must fulfill some requirements involving effective gelation, easy synthesis, low cost, and recyclable for reuse. However, beyond those, the ability of gelation for a broad-range oil phase without selectivity is also important. However, most of the reported PSOGs have limitation in this respect thus far. In this paper, a new class of saturated 1-monoglyceride-derived organogelators can efficiently uptake almost all of the common fuel oils from water and gelate organic solvents with extremely low minimum gelation concentration (MGC). In addition, the oils in the gel and gelator molecules can be recovered quantitatively through simple vacuum distillation.

  5. Diverse Broad-Host-Range Plasmids from Freshwater Carry Few Accessory Genes

    PubMed Central

    Sen, Diya; Yano, Hirokazu; Bauer, Matthew L.; Rogers, Linda M.; Van der Auwera, Geraldine A.

    2013-01-01

    Broad-host-range self-transferable plasmids are known to facilitate bacterial adaptation by spreading genes between phylogenetically distinct hosts. These plasmids typically have a conserved backbone region and a variable accessory region that encodes host-beneficial traits. We do not know, however, how well plasmids that do not encode accessory functions can survive in nature. The goal of this study was to characterize the backbone and accessory gene content of plasmids that were captured from freshwater sources without selecting for a particular phenotype or cultivating their host. To do this, triparental matings were used such that the only required phenotype was the plasmid's ability to mobilize a nonconjugative plasmid. Based on complete genome sequences of 10 plasmids, only 5 carried identifiable accessory gene regions, and none carried antibiotic resistance genes. The plasmids belong to four known incompatibility groups (IncN, IncP-1, IncU, and IncW) and two potentially new groups. Eight of the plasmids were shown to have a broad host range, being able to transfer into alpha-, beta-, and gammaproteobacteria. Because of the absence of antibiotic resistance genes, we resampled one of the sites and compared the proportion of captured plasmids that conferred antibiotic resistance to their hosts with the proportion of such plasmids captured from the effluent of a local wastewater treatment plant. Few of the captured plasmids from either site encoded antibiotic resistance. A high diversity of plasmids that encode no or unknown accessory functions is thus readily found in freshwater habitats. The question remains how the plasmids persist in these microbial communities. PMID:24096417

  6. Stable encoding of sounds over a broad range of statistical parameters in the auditory cortex.

    PubMed

    Blackwell, Jennifer M; Taillefumier, Thibaud O; Natan, Ryan G; Carruthers, Isaac M; Magnasco, Marcelo O; Geffen, Maria N

    2016-03-01

    Natural auditory scenes possess highly structured statistical regularities, which are dictated by the physics of sound production in nature, such as scale-invariance. We recently identified that natural water sounds exhibit a particular type of scale invariance, in which the temporal modulation within spectral bands scales with the centre frequency of the band. Here, we tested how neurons in the mammalian primary auditory cortex encode sounds that exhibit this property, but differ in their statistical parameters. The stimuli varied in spectro-temporal density and cyclo-temporal statistics over several orders of magnitude, corresponding to a range of water-like percepts, from pattering of rain to a slow stream. We recorded neuronal activity in the primary auditory cortex of awake rats presented with these stimuli. The responses of the majority of individual neurons were selective for a subset of stimuli with specific statistics. However, as a neuronal population, the responses were remarkably stable over large changes in stimulus statistics, exhibiting a similar range in firing rate, response strength, variability and information rate, and only minor variation in receptive field parameters. This pattern of neuronal responses suggests a potentially general principle for cortical encoding of complex acoustic scenes: while individual cortical neurons exhibit selectivity for specific statistical features, a neuronal population preserves a constant response structure across a broad range of statistical parameters.

  7. High-Sensitivity, Broad-Range Vacuum Gauge Using Nanotubes for Micromachined Cavities

    NASA Technical Reports Server (NTRS)

    Manohara, Harish; Kaul, Anupama B.

    2011-01-01

    A broad-range vacuum gauge has been created by suspending a single-walled carbon nanotube (SWNT) (metallic or semiconducting) in a Schottky diode format or in a bridge conductor format, between two electrically charged mesas. SWNTs are highly sensitive to molecular collisions because of their extremely small diameters in the range of 1 to 3 nanometers. The measurement parameter will be the change in conductivity of SWNT due to decreasing rate of molecular collisions as the pressure inside a chamber decreases. The rate of heat removal approaches a saturation limit as the mean free path (m.f.p.) lengths of molecules increase due to decreasing pressure. Only those sensing elements that have a long relaxation time can produce a measureable response when m.f.p. of molecules increases (or time between two consecutive collisions increases). A suspended SWNT offers such a capability because of its one-dimensional nature and ultrasmall diameter. In the initial approach, similar architecture was used as that of a SWNT-Schottky diode that has been developed at JPL, and has its changing conductivity measured as the test chamber is pumped down from atmospheric pressure to high vacuum (10(exp -7) Torr). Continuous response of decreasing conductivity has been measured as a function of decreasing pressure (SWNT is a negative thermal coefficient material) from atmosphere to less than 10(exp -6) Torr. A measureable current change in the hundreds of nA range has been recorded in the 10(exp -6) Torr regime.

  8. Broad range of 2050 warming from an observationally constrained large climate model ensemble

    NASA Astrophysics Data System (ADS)

    Rowlands, Daniel J.; Frame, David J.; Ackerley, Duncan; Aina, Tolu; Booth, Ben B. B.; Christensen, Carl; Collins, Matthew; Faull, Nicholas; Forest, Chris E.; Grandey, Benjamin S.; Gryspeerdt, Edward; Highwood, Eleanor J.; Ingram, William J.; Knight, Sylvia; Lopez, Ana; Massey, Neil; McNamara, Frances; Meinshausen, Nicolai; Piani, Claudio; Rosier, Suzanne M.; Sanderson, Benjamin M.; Smith, Leonard A.; Stone, Dáithí A.; Thurston, Milo; Yamazaki, Kuniko; Hiro Yamazaki, Y.; Allen, Myles R.

    2012-04-01

    Incomplete understanding of three aspects of the climate system--equilibrium climate sensitivity, rate of ocean heat uptake and historical aerosol forcing--and the physical processes underlying them lead to uncertainties in our assessment of the global-mean temperature evolution in the twenty-first century. Explorations of these uncertainties have so far relied on scaling approaches, large ensembles of simplified climate models, or small ensembles of complex coupled atmosphere-ocean general circulation models which under-represent uncertainties in key climate system properties derived from independent sources. Here we present results from a multi-thousand-member perturbed-physics ensemble of transient coupled atmosphere-ocean general circulation model simulations. We find that model versions that reproduce observed surface temperature changes over the past 50 years show global-mean temperature increases of 1.4-3K by 2050, relative to 1961-1990, under a mid-range forcing scenario. This range of warming is broadly consistent with the expert assessment provided by the Intergovernmental Panel on Climate Change Fourth Assessment Report, but extends towards larger warming than observed in ensembles-of-opportunity typically used for climate impact assessments. From our simulations, we conclude that warming by the middle of the twenty-first century that is stronger than earlier estimates is consistent with recent observed temperature changes and a mid-range `no mitigation' scenario for greenhouse-gas emissions.

  9. Enhanced primers for amplification of DNA barcodes from a broad range of marine metazoans.

    PubMed

    Lobo, Jorge; Costa, Pedro M; Teixeira, Marcos A L; Ferreira, Maria S G; Costa, Maria H; Costa, Filipe O

    2013-09-10

    Building reference libraries of DNA barcodes is relatively straightforward when specifically designed primers are available to amplify the COI-5P region from a relatively narrow taxonomic group (e.g. single class or single order). DNA barcoding marine communities have been comparatively harder to accomplish due to the broad taxonomic diversity and lack of consistently efficient primers. Although some of the so-called "universal" primers have been relatively successful, they still fail to amplify COI-5P of many marine animal groups, while displaying random success even among species within each group. Here we propose a new pair of primers designed to enhance amplification of the COI-5P region in a wide range of marine organisms. Amplification tests conducted on a wide range of marine animal taxa, rendered possible the first-time sequencing of DNA barcodes from eight separated phyla (Annelida, Arthropoda, Chordata, Cnidaria, Echinodermata, Mollusca, Nemertea and Platyhelminthes), comprising a total of 14 classes, 28 orders, 57 families, 68 genus and 76 species. These primers demonstrated to be highly cost-effective, which is of key importance for DNA barcoding procedures, such as for building comprehensive DNA barcode libraries of marine communities, where the processing of a large numbers of specimens from a wide variety of marine taxa is compulsory.

  10. Enhanced primers for amplification of DNA barcodes from a broad range of marine metazoans

    PubMed Central

    2013-01-01

    Background Building reference libraries of DNA barcodes is relatively straightforward when specifically designed primers are available to amplify the COI-5P region from a relatively narrow taxonomic group (e.g. single class or single order). DNA barcoding marine communities have been comparatively harder to accomplish due to the broad taxonomic diversity and lack of consistently efficient primers. Although some of the so-called “universal” primers have been relatively successful, they still fail to amplify COI-5P of many marine animal groups, while displaying random success even among species within each group. Here we propose a new pair of primers designed to enhance amplification of the COI-5P region in a wide range of marine organisms. Results Amplification tests conducted on a wide range of marine animal taxa, rendered possible the first–time sequencing of DNA barcodes from eight separated phyla (Annelida, Arthropoda, Chordata, Cnidaria, Echinodermata, Mollusca, Nemertea and Platyhelminthes), comprising a total of 14 classes, 28 orders, 57 families, 68 genus and 76 species. Conclusions These primers demonstrated to be highly cost-effective, which is of key importance for DNA barcoding procedures, such as for building comprehensive DNA barcode libraries of marine communities, where the processing of a large numbers of specimens from a wide variety of marine taxa is compulsory. PMID:24020880

  11. Noradrenaline transmission reducing drugs may protect against a broad range of diseases.

    PubMed

    Fitzgerald, P J

    2015-04-01

    1 A growing body of evidence suggests that the signalling molecule, noradrenaline (NA), plays a pathophysiological role in a broad range of psychiatric, neurological and peripheral disorders. Both preclinical and clinical data suggest that elevated NA signalling may be involved in the aetiology of major diseases such as depression, Alzheimer's disease and diabetes mellitus. 2 The molecular pathways by which NA may cause the manifestation of disease remain poorly understood, although they may include G protein-coupled receptor modulation of the Ras/MAP kinase, Stat3 and PI3K pathways, among others. In both individual animals and humans, NA tone may be elevated largely due to genetics, but also because of the exposure to marked psychological stress or trauma, or other environmental factors. 3 As NA is involved in the 'fight or flight' response by the sympathetic nervous system, this transmitter may be elevated in a large number of organisms due to evolutionary selection of enhancing responses to immediate environmental dangers. Likewise, acetylcholine signalling by the parasympathetic ('rest and digest') nervous system may be relatively diminished. This putative autonomic imbalance may result in diminished engagement in homeostatic processes, resulting in the emergence and progression of a number of diseases throughout the body. 4 In this scenario, a large number of individuals may benefit from chronic use of pharmacological agents - such as clonidine, guanfacine, propranolol or prazosin - that diminish NA signalling throughout the body. If so, NA transmission lowering drugs may protect against a wide range of diseases.

  12. A new technique for the characterization of long-range tertiary contacts in large RNA molecules: insertion of a photolabel at a selected position in 16S rRNA within the Escherichia coli ribosome.

    PubMed Central

    Baranov, P V; Dokudovskaya, S S; Oretskaya, T S; Dontsova, O A; Bogdanov, A A; Brimacombe, R

    1997-01-01

    A new approach for inserting a photo-label at a selected position within the long ribosomal RNA molecules has been developed. The Escherichia coli 16S rRNA was cleaved at a single internucleotide bond, 1141-1142, with RNase H in the presence of a complementary chimeric oligonucleotide. 4-Thiouridine 5', 3'-diphosphate was ligated to the 3'-end of the 5'fragment at the cleavage site with T4 RNA ligase. The 16S rRNA fragments containing this added photo-reactive nucleotide were assembled together with total 30S ribosomal proteins into small ribosomal subunits. The ability of such 30S particles containing fragmented rRNA to form 70S ribosomes has been demonstrated previously. Crosslinks were induced within the 30S subunits by mild UV irradiation. The sites of crosslinking within the 16S rRNA were then analyzed using RNase H digestion and reverse transcription. Two crosslinks from the thio-nucleotide attached to nt C1141 of 16S rRNA were observed, namely to nt U1295 and G1272. These results are in agreement with the established proximity of helix 39 and 41 in the 3D structure of the 30S ribosomal subunit, as shown by other intra RNA crosslinking data. These data furthermore allow us to refine the structural arrangement of helices 41 and 39 relative to one another. PMID:9171076

  13. A broad-range method to detect genomic DNA of multiple pathogenic bacteria based on the aggregation strategy of gold nanorods.

    PubMed

    Wang, Xiaohui; Li, Yuan; Wang, Jidong; Wang, Quanli; Xu, Lijuan; Du, Juan; Yan, Shaoduo; Zhou, Yong; Fu, Qiuxia; Wang, Yingli; Zhan, Linsheng

    2012-09-21

    It remains challenging to detect unknown pathogenic bacteria in diagnostic, clinical and environmental fields. This work describes the approach to the development of a sensitive, broad-range genosensing assay targeting the conserved 16S rDNA region existing in most bacteria, by monitoring the aggregation level of gold nanorods (GNRs)-based nanoprobes through their localized surface plasmon resonance (LSPR) property. In the quantitative detection of artificial sequence, the limit of detection (LOD) of such a bioassay is demonstrated to reach the 5 pM level. This pair of universal GNRs-based nanoprobes can further identify at least 6 species of bacteria that were most prevalent in platelet concentrates (PCs) and have no cross-reaction with other pathogens. Moreover, it also exhibits higher sensitivity than other broad-range methods in analysing Serratia marcescens-spiked PCs. Therefore, the presented strategy not only provides a novel and effective DNA analysis method to detect multiple bacterial contaminations in PCs, but also opens up possibilities for its future use of detecting unknown bacteria in other systems, such as food and water, even at ultralow levels.

  14. Broad-Range Antiviral Activity of Hydrogen Sulfide Against Highly Pathogenic RNA Viruses

    PubMed Central

    Bazhanov, Nikolay; Escaffre, Olivier; Freiberg, Alexander N.; Garofalo, Roberto P.; Casola, Antonella

    2017-01-01

    Hydrogen sulfide is an important endogenous mediator that has been the focus of intense investigation in the past few years, leading to the discovery of its role in vasoactive, cytoprotective and anti-inflammatory responses. Recently, we made a critical observation that H2S also has a protective role in paramyxovirus infection by modulating inflammatory responses and viral replication. In this study we tested the antiviral and anti-inflammatory activity of the H2S slow-releasing donor GYY4137 on enveloped RNA viruses from Ortho-, Filo-, Flavi- and Bunyavirus families, for which there is no FDA-approved vaccine or therapeutic available, with the exception of influenza. We found that GYY4137 significantly reduced replication of all tested viruses. In a model of influenza infection, GYY4137 treatment was associated with decreased expression of viral proteins and mRNA, suggesting inhibition of an early step of replication. The antiviral activity coincided with the decrease of viral-induced pro-inflammatory mediators and viral-induced nuclear translocation of transcription factors from Nuclear Factor (NF)-kB and Interferon Regulatory Factor families. In conclusion, increasing cellular H2S is associated with significant antiviral activity against a broad range of emerging enveloped RNA viruses, and should be further explored as potential therapeutic approach in relevant preclinical models of viral infections. PMID:28106111

  15. Path dependence of climate and carbon cycle response over a broad range of cumulative carbon emissions

    NASA Astrophysics Data System (ADS)

    Herrington, T.; Zickfeld, K.

    2014-06-01

    Recent studies have demonstrated the proportional relationship between global warming and cumulative carbon emissions, yet the robustness of this relationship has not been tested over a broad range of cumulative emissions and emission rates. This study explores the path dependence of the climate and carbon cycle response using an Earth System model of intermediate complexity forced with 24 idealized emissions scenarios across five cumulative emission groups (1275-5275 GtC) with varying rates of emission. We find the century-scale climate and carbon cycle response after cessation of emissions to be approximately independent of emission pathway for all cumulative emission levels considered. The ratio of global mean temperature change to cumulative emissions - referred to as the transient climate response to cumulative emissions (TCRE) - is found to be constant for cumulative emissions lower than ~1500 GtC, but to decline with higher cumulative emissions. The TCRE is also found to decrease with increasing emission rate. The response of Arctic sea ice is found to be approximately proportional to cumulative emissions, while the response of the Atlantic meridional overturning circulation (AMOC) does not scale linearly with cumulative emissions, as its peak response is strongly dependent on emission rate. Ocean carbon uptake weakens with increasing cumulative emissions, while land carbon uptake displays non-monotonic behavior, increasing up to a cumulative emission threshold of ~2000 GtC and then declining.

  16. Path independence of climate and carbon cycle response over a broad range of cumulative carbon emissions

    NASA Astrophysics Data System (ADS)

    Herrington, T.; Zickfeld, K.

    2014-11-01

    Recent studies have identified an approximately proportional relationship between global warming and cumulative carbon emissions, yet the robustness of this relationship has not been tested over a broad range of cumulative emissions and emission rates. This study explores the path dependence of the climate and carbon cycle response using an Earth system model of intermediate complexity forced with 24 idealized emissions scenarios across five cumulative emission groups (1275-5275 Gt C) with varying rates of emission. We find the century-scale climate and carbon cycle response after cessation of emissions to be approximately independent of emission pathway for all cumulative emission levels considered. The ratio of global mean temperature change to cumulative emissions - referred to as the transient climate response to cumulative carbon emissions (TCRE) - is found to be constant for cumulative emissions lower than ∼1500 Gt C but to decline with higher cumulative emissions. The TCRE is also found to decrease with increasing emission rate. The response of Arctic sea ice is found to be approximately proportional to cumulative emissions, while the response of the Atlantic Meridional Overturning Circulation does not scale linearly with cumulative emissions, as its peak response is strongly dependent on emission rate. Ocean carbon uptake weakens with increasing cumulative emissions, while land carbon uptake displays non-monotonic behavior, increasing up to a cumulative emission threshold of ∼2000 Gt C and then declining.

  17. A Lactobacillus plantarum Esterase Active on a Broad Range of Phenolic Esters

    PubMed Central

    Esteban-Torres, María; Landete, José María; Reverón, Inés; Santamaría, Laura; de las Rivas, Blanca

    2015-01-01

    Lactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments. PMID:25746986

  18. A Lactobacillus plantarum esterase active on a broad range of phenolic esters.

    PubMed

    Esteban-Torres, María; Landete, José María; Reverón, Inés; Santamaría, Laura; de las Rivas, Blanca; Muñoz, Rosario

    2015-05-01

    Lactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Oxidative Stress and Mitochondrial Dysfunction across Broad-Ranging Pathologies: Toward Mitochondria-Targeted Clinical Strategies

    PubMed Central

    d'Ischia, Marco; Gadaleta, Maria Nicola; Pallardó, Federico V.; Petrović, Sandra; Tiano, Luca; Zatterale, Adriana

    2014-01-01

    Beyond the disorders recognized as mitochondrial diseases, abnormalities in function and/or ultrastructure of mitochondria have been reported in several unrelated pathologies. These encompass ageing, malformations, and a number of genetic or acquired diseases, as diabetes and cardiologic, haematologic, organ-specific (e.g., eye or liver), neurologic and psychiatric, autoimmune, and dermatologic disorders. The mechanistic grounds for mitochondrial dysfunction (MDF) along with the occurrence of oxidative stress (OS) have been investigated within the pathogenesis of individual disorders or in groups of interrelated disorders. We attempt to review broad-ranging pathologies that involve mitochondrial-specific deficiencies or rely on cytosol-derived prooxidant states or on autoimmune-induced mitochondrial damage. The established knowledge in these subjects warrants studies aimed at elucidating several open questions that are highlighted in the present review. The relevance of OS and MDF in different pathologies may establish the grounds for chemoprevention trials aimed at compensating OS/MDF by means of antioxidants and mitochondrial nutrients. PMID:24876913

  20. Tuning the Polarization State of Light over a Broad Frequency Range with Metasurfaces

    NASA Astrophysics Data System (ADS)

    Wang, Mu; Jiang, Shang-Chi; Wang, Zheng-Han; Xiong, Xiang; Peng, Ru-Wen; Nanjing University Team

    Controlling the polarization state, the transmission direction and the phase of light within a confined space is an important issue in optics. By integrating metallic metastructure and dielectric interlayer, it is possible to realize the dispersion-free broadband device on sub-wavelength scale, where the strong response of the metallic structures helps to decrease the device size while the dielectric interlayer helps to eliminate the dispersion simultaneously in both the amplitude and the phase difference of the reflected/transmitted light. As an examples to apply this concept, a broadband quarter-wave plate and a half-wave plate are experimentally demonstrated. By carefully selecting the structural parameters, the polarization state of light can be freely tuned across a broad frequency range, and all of the polarization states on the Poincaré sphere can be realized dispersion free. Some contents of this talk can be found in the following references: [1] S.-C. Jiang, et al., High-efficiency generation of circularly polarized light via symmetry-induced anomalous reflection,Physical Review B 91, 125421 (2015), [2] S.-C. Jiang, et al., Controlling the Polarization State of Light with a Dispersion-Free Metastructure, Physical Review X 4, 021026 (2014), [3] X. Xiong, et al., Metallic stereostructured layer: an approach for broadband polarization state manipulation,Applied Physics Letters 105, 201105 (2014).

  1. The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range.

    PubMed

    Korshunova, Y O; Eide, D; Clark, W G; Guerinot, M L; Pakrasi, H B

    1999-05-01

    The molecular basis for the transport of manganese across membranes in plant cells is poorly understood. We have found that IRT1, an Arabidopsis thaliana metal ion transporter, can complement a mutant Saccharomyces cerevisiae strain defective in high-affinity manganese uptake (smf1 delta). The IRT1 protein has previously been identified as an iron transporter. The current studies demonstrated that IRT1, when expressed in yeast, can transport manganese as well. This manganese uptake activity was inhibited by cadmium, iron(II) and zinc, suggesting that IRT1 can transport these metals. The IRT1 cDNA also complements a zinc uptake-deficient yeast mutant strain (zrt1zrt2), and IRT1-dependent zinc transport in yeast cells is inhibited by cadmium, copper, cobalt and iron(III). However, IRT1 did not complement a copper uptake-deficient yeast mutant (ctr1), implying that this transporter is not involved in the uptake of copper in plant cells. The expression of IRT1 is enhanced in A. thaliana plants grown under iron deficiency. Under these conditions, there were increased levels of root-associated manganese, zinc and cobalt, suggesting that, in addition to iron, IRT1 mediates uptake of these metals into plant cells. Taken together, these data indicate that the IRT1 protein is a broad-range metal ion transporter in plants.

  2. Streptolysin S of Streptococcus anginosus exhibits broad-range hemolytic activity.

    PubMed

    Asam, Daniela; Mauerer, Stefanie; Spellerberg, Barbara

    2015-04-01

    Streptococcus anginosus is a commensal of mucous membranes and an emerging human pathogen. Some strains, including the type strain, display a prominent β-hemolytic phenotype. A gene cluster (sag), encoding a variant of streptolysin S (SLS) has recently been identified as the genetic background for β-hemolysin production in S. anginosus. In this study, we further characterized the hemolytic and cytolytic activity of the S. anginosus hemolysin in comparison with other streptococcal hemolysins. The results indicate that SLS of S. anginosus is a broad-range hemolysin able to lyse erythrocytes of different species, including horse, bovine, rabbit and even chicken. The hemolytic activity is temperature dependent, and a down-regulation of the hemolysin expression is induced in the presence of high glucose levels. Survival assays indicate that in contrast to other streptococcal species, S. anginosus does not require SLS for survival in the presence of human granulocytes. Cross-complementation studies using the sagB and sagD genes of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis demonstrated functional similarities to the S. anginosus SLS. Nevertheless, distinct differences to other streptolysin S variants were noted and provide further insights into the molecular mechanisms of SLS pathogen host interactions.

  3. Accelerating the introduction of HTS products for a broad range of electric power and industrial applications

    NASA Astrophysics Data System (ADS)

    Eaton, Russell

    2002-01-01

    The Department of Energy (DOE), as part of its Superconductivity Program for Electric Systems, is successfully pursuing the development of electric power and industrial devices, incorporating significant high-temperature superconducting (HTS) components or subsystems, through its innovative Superconducting Partnership Initiative (SPI). The objective of the SPI is to accelerate the commercial introduction of the HTS products for a broad range of electric power and industrial applications. DOE's approach to accomplishing the SPI objective is to support cost shared projects carried out by industry led teams. DOE will fund projects to develop HTS devices that are either in (1) the research and development stage (Phase 1), (2) the pre-commercialization stage (Phase II), or (3) the commercial entry stage (Phase III). DOE's industry partners must contribute at least half a project's costs. These teams will include capabilities needed to develop the device as well as to develop the business plan for the commercial product introduction. DOE's partners consist of vertically integrated teams consisting of equipment manufacturers, HTS wire and coil suppliers, national laboratories, and end users, primarily utilities. These partners carry out the multi-year technology development efforts, consisting generally of design, construction, and testing of the HTS system. Finally, commercialization of HTS products will be discussed primarily in terms of benefits these products will have over competing products based upon conventional conductors and the critical need for affordable, practical HTS materials and conductors for these applications. .

  4. Personal glucose meters for detection and quantification of a broad range of analytes

    DOEpatents

    Lu, Yi; Xiang, Yu

    2015-02-03

    A general methodology for the development of highly sensitive and selective sensors that can achieve portable, low-cost and quantitative detection of a broad range of targets using only a personal glucose meter (PGM) is disclosed. The method uses recognition molecules that are specific for a target agent, enzymes that can convert an enzyme substrate into glucose, and PGM. Also provided are sensors, which can include a solid support to which is attached a recognition molecule that permits detection of a target agent, wherein the recognition molecule specifically binds to the target agent in the presence of the target agent but not significantly to other agents as well as an enzyme that can catalyze the conversion of a substance into glucose, wherein the enzyme is attached directly or indirectly to the recognition molecule, and wherein in the presence of the target agent the enzyme can convert the substance into glucose. The disclosed sensors can be part of a lateral flow device. Methods of using such sensors for detecting target agents are also provided.

  5. Construction and characterization of regulated L-arabinose-inducible broad host range expression vectors in Xanthomonas.

    PubMed

    Sukchawalit, R; Vattanaviboon, P; Sallabhan, R; Mongkolsuk, S

    1999-12-15

    Several versions of broad host range (BHR), L-arabinose-inducible expression vectors were constructed. These expression vectors were based on a high copy number BHR pBBR1MCS-4 replicon that could replicate in both enteric and non-enteric Gram-negative bacteria. Two versions of expression cassettes containing multiple cloning sites either with or without a ribosome binding site were placed under transcriptional control of the Escherichia coli BAD promoter and araC gene. Three versions of vectors containing ampicillin or kanamycin or tetracycline resistance genes as selectable markers were constructed. In all six new L-arabinose-inducible BHR expression vectors containing many unique cloning sites, selectable markers were made to facilitate cloning and expression of genes in various Gram-negative bacteria. A Tn9 chloramphenicol acetyl transferase (cat) gene was cloned into an expression vector, resulting in pBBad18Acat that was used to establish optimal expression conditions (addition of 0.02% L-arabinose to mid-exponential phase cells for at least 1 h) in a Xanthomonas campestris pv. phaseoli. Comparison of the Cat enzyme activities between uninduced and a 180-min L-arabinose-induced culture showed a greater than 150-fold increased Cat specific activity. In addition, L-arabinose induction of exponential phase cells harboring pBBad18Acat gave a higher amount of Cat than similarly treated stationary phase cells. The usefulness of the expression vector was also demonstrated in both enteric and non-enteric Gram-negative bacteria.

  6. Testing and Validation of High Density Resequencing Microarray for Broad Range Biothreat Agents Detection

    PubMed Central

    Leski, Tomasz A.; Lin, Baochuan; Malanoski, Anthony P.; Wang, Zheng; Long, Nina C.; Meador, Carolyn E.; Barrows, Brian; Ibrahim, Sofi; Hardick, Justin P.; Aitichou, Mohamed; Schnur, Joel M.; Tibbetts, Clark; Stenger, David A.

    2009-01-01

    Rapid and effective detection and identification of emerging microbiological threats and potential biowarfare agents is very challenging when using traditional culture-based methods. Contemporary molecular techniques, relying upon reverse transcription and/or polymerase chain reaction (RT-PCR/PCR) provide a rapid and effective alternative, however, such assays are generally designed and optimized to detect only a limited number of targets, and seldom are capable of differentiation among variants of detected targets. To meet these challenges, we have designed a broad-range resequencing pathogen microarray (RPM) for detection of tropical and emerging infectious agents (TEI) including biothreat agents: RPM-TEI v 1.0 (RPM-TEI). The scope of the RPM-TEI assay enables detection and differential identification of 84 types of pathogens and 13 toxin genes, including most of the class A, B and C select agents as defined by the Centers for Disease Control and Prevention (CDC, Atlanta, GA). Due to the high risks associated with handling these particular target pathogens, the sensitivity validation of the RPM-TEI has been performed using an innovative approach, in which synthetic DNA fragments are used as templates for testing the assay's limit of detection (LOD). Assay specificity and sensitivity was subsequently confirmed by testing with full-length genomic nucleic acids of selected agents. The LOD for a majority of the agents detected by RPM-TEI was determined to be at least 104 copies per test. Our results also show that the RPM-TEI assay not only detects and identifies agents, but is also able to differentiate near neighbors of the same agent types, such as closely related strains of filoviruses of the Ebola Zaire group, or the Machupo and Lassa arenaviruses. Furthermore, each RPM-TEI assay results in specimen-specific agent gene sequence information that can be used to assess pathogenicity, mutations, and virulence markers, results that are not generally available from

  7. Forest response to elevated CO2 is conserved across a broad range of productivity

    PubMed Central

    Norby, Richard J.; DeLucia, Evan H.; Gielen, Birgit; Calfapietra, Carlo; Giardina, Christian P.; King, John S.; Ledford, Joanne; McCarthy, Heather R.; Moore, David J. P.; Ceulemans, Reinhart; De Angelis, Paolo; Finzi, Adrien C.; Karnosky, David F.; Kubiske, Mark E.; Lukac, Martin; Pregitzer, Kurt S.; Scarascia-Mugnozza, Giuseppe E.; Schlesinger, William H.; Oren, Ram

    2005-01-01

    Climate change predictions derived from coupled carbon-climate models are highly dependent on assumptions about feedbacks between the biosphere and atmosphere. One critical feedback occurs if C uptake by the biosphere increases in response to the fossil-fuel driven increase in atmospheric [CO2] (“CO2 fertilization”), thereby slowing the rate of increase in atmospheric [CO2]. Carbon exchanges between the terrestrial biosphere and atmosphere are often first represented in models as net primary productivity (NPP). However, the contribution of CO2 fertilization to the future global C cycle has been uncertain, especially in forest ecosystems that dominate global NPP, and models that include a feedback between terrestrial biosphere metabolism and atmospheric [CO2] are poorly constrained by experimental evidence. We analyzed the response of NPP to elevated CO2 (≈550 ppm) in four free-air CO2 enrichment experiments in forest stands. We show that the response of forest NPP to elevated [CO2] is highly conserved across a broad range of productivity, with a stimulation at the median of 23 ± 2%. At low leaf area indices, a large portion of the response was attributable to increased light absorption, but as leaf area indices increased, the response to elevated [CO2] was wholly caused by increased light-use efficiency. The surprising consistency of response across diverse sites provides a benchmark to evaluate predictions of ecosystem and global models and allows us now to focus on unresolved questions about carbon partitioning and retention, and spatial variation in NPP response caused by availability of other growth limiting resources. PMID:16330779

  8. Monoclonal antibodies to VP1 recognize a broad range of enteroviruses.

    PubMed

    Miao, Lynn Yihong; Pierce, Christina; Gray-Johnson, Jennifer; DeLotell, Jill; Shaw, Carl; Chapman, Nate; Yeh, Elaine; Schnurr, David; Huang, Yung T

    2009-10-01

    Enteroviruses (EVs) are common seasonal viruses that are associated with a variety of diseases. High-quality monoclonal antibodies (MAbs) are needed to improve the accuracy of EV diagnosis in clinical laboratories. In the present study, the full-length VP1 genes of poliovirus 1 (Polio 1) and coxsackievirus B3 (Cox B3) were cloned, and the encoded proteins were expressed and used as antigens in an attempt to raise broad-spectrum MAbs to EVs. Two pan-EV MAbs were isolated: one raised against Polio 1 VP1 and the other against Cox B3 VP1. The binding sites of both pan-EV MAbs were mapped to an amino acid sequence within a conserved region in the N terminus of Polio 1 VP1 by peptide and competition enzyme-linked immunosorbent assay. Two additional MAbs, an EV70-specific MAb and an EV71/Cox A16-bispecific MAb, developed against EV70 and 71 VP1 proteins, were pooled with the two pan-EV MAbs (pan-EV MAb mix) and tested for their sensitivity and specificity in the staining of various virus-infected cells. The pan-EV MAb mix detected all 40 prototype EVs tested and showed no cross-reactivity to 18 different non-EV human viruses. Compared with two commercially available EV tests, the pan-EV MAb mix exhibited higher specificity than one test and broader spectrum reactivity than the other. Thus, our study demonstrates that full-length Polio 1 VP1 and Cox B3 VP1 can serve as effective antigens for developing a pan-EV MAb and that the pan-EV MAb mix can be used for the laboratory diagnosis of a wide range of EV infections.

  9. Universal scaling law for the condensation energy across a broad range of superconductor classes

    NASA Astrophysics Data System (ADS)

    Kim, J. S.; Tam, G. N.; Stewart, G. R.

    2015-12-01

    One of the goals in understanding any new class of superconductors is to search for commonalities with other known superconductors. The present work investigates the superconducting condensation energy, U , in the iron based superconductors (IBSs), and compares their U with a broad range of other distinct classes of superconductor, including conventional BCS elements and compounds and the unconventional heavy fermion, S r2Ru O4 ,L i0.1ZrNCl ,κ -(BEDT-TTF)2Cu (NCS )2 , and optimally doped cuprate superconductors. Surprisingly, both the magnitude and Tc dependence (U ∝Tc3.4 ±0.2 ) of U are—contrary to the previously observed behavior of the specific heat discontinuity at Tc, Δ C —quite similar in the IBS and BCS materials for Tc>1.4 K. In contrast, the heavy fermion superconductors' U vs Tc are strongly (up to a factor of 100) enhanced above the IBS/BCS while the cuprate superconductors' U are strongly (factor of 8) reduced. However, scaling of U with the specific heat γ (or Δ C ) brings all the superconductors investigated onto one universal dependence upon Tc. This apparent universal scaling U / γ ∝Tc2 for all superconductor classes investigated, both weak and strong coupled and both conventional and unconventional, links together extremely disparate behaviors over almost seven orders of magnitude for U and almost three orders of magnitude for Tc. Since U has not yet been explicitly calculated beyond the weak coupling limit, the present results can help direct theoretical efforts into the medium and strong coupling regimes.

  10. Forest response to elevated CO2 is conserved across a broad range of productivity.

    PubMed

    Norby, Richard J; Delucia, Evan H; Gielen, Birgit; Calfapietra, Carlo; Giardina, Christian P; King, John S; Ledford, Joanne; McCarthy, Heather R; Moore, David J P; Ceulemans, Reinhart; De Angelis, Paolo; Finzi, Adrien C; Karnosky, David F; Kubiske, Mark E; Lukac, Martin; Pregitzer, Kurt S; Scarascia-Mugnozza, Giuseppe E; Schlesinger, William H; Oren, Ram

    2005-12-13

    Climate change predictions derived from coupled carbon-climate models are highly dependent on assumptions about feedbacks between the biosphere and atmosphere. One critical feedback occurs if C uptake by the biosphere increases in response to the fossil-fuel driven increase in atmospheric [CO(2)] ("CO(2) fertilization"), thereby slowing the rate of increase in atmospheric [CO(2)]. Carbon exchanges between the terrestrial biosphere and atmosphere are often first represented in models as net primary productivity (NPP). However, the contribution of CO(2) fertilization to the future global C cycle has been uncertain, especially in forest ecosystems that dominate global NPP, and models that include a feedback between terrestrial biosphere metabolism and atmospheric [CO(2)] are poorly constrained by experimental evidence. We analyzed the response of NPP to elevated CO(2) ( approximately 550 ppm) in four free-air CO(2) enrichment experiments in forest stands. We show that the response of forest NPP to elevated [CO(2)] is highly conserved across a broad range of productivity, with a stimulation at the median of 23 +/- 2%. At low leaf area indices, a large portion of the response was attributable to increased light absorption, but as leaf area indices increased, the response to elevated [CO(2)] was wholly caused by increased light-use efficiency. The surprising consistency of response across diverse sites provides a benchmark to evaluate predictions of ecosystem and global models and allows us now to focus on unresolved questions about carbon partitioning and retention, and spatial variation in NPP response caused by availability of other growth limiting resources.

  11. Theoretical Study of Radiation from a Broad Range of Impurity Ions for Magnetic Fusion Diagnostics

    SciTech Connect

    Safronova, Alla

    2014-03-14

    Spectroscopy of radiation emitted by impurities plays an important role in the study of magnetically confined fusion plasmas. The measurements of these impurities are crucial for the control of the general machine conditions, for the monitoring of the impurity levels, and for the detection of various possible fault conditions. Low-Z impurities, typically present in concentrations of 1%, are lithium, beryllium, boron, carbon, and oxygen. Some of the common medium-Z impurities are metals such as iron, nickel, and copper, and high-Z impurities, such as tungsten, are present in smaller concentrations of 0.1% or less. Despite the relatively small concentration numbers, the aforementioned impurities might make a substantial contribution to radiated power, and also influence both plasma conditions and instruments. A detailed theoretical study of line radiation from impurities that covers a very broad spectral range from less than 1 Å to more than 1000 Å has been accomplished and the results were applied to the LLNL Electron Beam Ion Trap (EBIT) and the Sustained Spheromak Physics Experiment (SSPX) and to the National Spherical Torus Experiment (NSTX) at Princeton. Though low- and medium-Z impurities were also studied, the main emphasis was made on the comprehensive theoretical study of radiation from tungsten using different state-of-the-art atomic structure codes such as Relativistic Many-Body Perturbation Theory (RMBPT). The important component of this research was a comparison of the results from the RMBPT code with other codes such as the Multiconfigurational Hartree–Fock developed by Cowan (COWAN code) and the Multiconfiguration Relativistic Hebrew University Lawrence Atomic Code (HULLAC code), and estimation of accuracy of calculations. We also have studied dielectronic recombination, an important recombination process for fusion plasma, for variety of highly and low charged tungsten ions using COWAN and HULLAC codes. Accurate DR rate coefficients are needed for

  12. Broad-Host Range Vector-Particle: Gene Transfer Particles From Thermal Vents

    NASA Astrophysics Data System (ADS)

    Chiura, H. X.; Nakamura, K.; Fukazawa, Y.; Nakata, D.; Tomaru, A.; Okita, N.; Hoaki, T.

    2002-12-01

    Viruses or virus-like particles (VLPs) are common in aquatic ecosystems, however, VLP-host interactions and its commitments to gene transfer in the environment is yet unclear. We have proposed that at least some of the widely distributed VLPs could be general gene transfer agents among a wide range of microbial host cells, and might function as a universal vector (1-4). To elucidate such a broad host range gene transfer mediated by "VLP", the sampling site was extended to the hyper hydrothermal vent, and boring cores. VLP (v) and cell (b) abundances per ml water samples from drilling holes of Suiyo seamount were: APSK04 (28°34.303'N, 140°38.618'E, 1385 m deep, 21°C, b = 8.26 *E^{6}, v = 6.03 x 10^{6}); APSK07 (28°34.299'N, 140°38.690'E, 1386 m deep, 250.5°C, b = 5.33 \\times 104, v = 2.52 \\times 104); a natural vent near APSK05 (28°34.322'N, 140°38.594'E, 1382 m deep, 304.7°C, b = 3.23 x 10^{4}, v = 1.85 x 10^{4}). A boring core sample was obtained from APSK06 (28°34.313'N, 140°38.617', 1386 m deep), from which a hyper thermophilic Archaean, Thermococcus kodakaraensis was successfully cultivated in sulphur supplemented medium between 70 and 90°C. VLP production was observed from T. kodakaraensis, whose VLP (v) and cell (b) abundances per ml at 480 h culture at 70°C were: b = 3.61 *E^{9}, v = 3.46 *E^{9}. Transduction experiment at multiplicity of infection of ca 0.2 using particles from APSK07 and T. kodakaraensis showed a plate efficiency on recipient Escherichia coli AB1157 by ca 72 % and ca 89 % regardless of UV treatment of the particle. Gene transfer frequency of APSK07 particle was (x 10^{-5} cfu/particle) between 2.4 and 0.92, and that of T. kodakaraensis particle was between x 10^{-4} and x 10^{-5}$ cfu/particle. These findings suggest the non-specific gene transfer by such particles may be a ubiquitous event in the natural environment. Such gene transfer particles may have mediated gene flux among phylogenetically diverse microbial

  13. Utility of 16S rRNA PCR performed on clinical specimens in patient management.

    PubMed

    Akram, A; Maley, M; Gosbell, I; Nguyen, T; Chavada, R

    2017-04-01

    Broad-range 16S rRNA PCR can be used for the detection and identification of bacteria from clinical specimens in patients for whom there is a high suspicion of infection and cultures are negative. The aims of this study were (1) to compare 16S rRNA PCR results with microbiological culture results, (2) to assess the utility of 16S rRNA PCR with regard to antimicrobial therapy, and (3) to compare the yield of 16S rRNA PCR for different types of clinical specimen and to perform a cost analysis of the test. A retrospective study was performed on different clinical specimens which had 16S performed over 3 years (2012-2015). Standard microbiological cultures were performed on appropriate media, as per the laboratory protocol. Patient clinical and microbiological data were obtained from the electronic medical records and laboratory information system, respectively. 16S rRNA PCR was performed in a reference laboratory using a validated method for amplification and sequencing. The outcomes assessed were the performance of 16S rRNA PCR, change of antimicrobials (rationalization, cessation, or addition), and duration of therapy. Concordance of 16S rRNA PCR with bacterial cultures was also determined for tissue specimens. Thirty-two patients were included in the study, for whom an equal number of specimens (n=32) were sent for 16S rRNA PCR. 16S rRNA PCR could identify an organism in 10 of 32 cases (31.2%), of which seven were culture-positive and three were culture-negative. The sensitivity was 58% (confidence interval (CI) 28.59-83.5%) and specificity was 85% (CI 61.13-96%), with a positive predictive value of 70% (CI 35.3-91.9%) and negative predictive value of 77.2% (CI 54.17-91.3%). Antimicrobial therapy was rationalized after 16S rRNA PCR results in five patients (15.6%) and was ceased in four based on negative results (12.5%). Overall the 16S rRNA PCR result had an impact on antimicrobial therapy in 28% of patients (9/32). The highest concordance of 16S rRNA PCR with

  14. Analysis of bacterial DNA in synovial tissue of Tunisian patients with reactive and undifferentiated arthritis by broad-range PCR, cloning and sequencing

    PubMed Central

    Siala, Mariam; Jaulhac, Benoit; Gdoura, Radhouane; Sibilia, Jean; Fourati, Hela; Younes, Mohamed; Baklouti, Sofien; Bargaoui, Naceur; Sellami, Slaheddine; Znazen, Abir; Barthel, Cathy; Collin, Elody; Hammami, Adnane; Sghir, Abdelghani

    2008-01-01

    Introduction Bacteria and/or their antigens have been implicated in the pathogenesis of reactive arthritis (ReA). Several studies have reported the presence of bacterial antigens and nucleic acids of bacteria other than those specified by diagnostic criteria for ReA in joint specimens from patients with ReA and various arthritides. The present study was conducted to detect any bacterial DNA and identify bacterial species that are present in the synovial tissue of Tunisian patients with reactive arthritis and undifferentiated arthritis (UA) using PCR, cloning and sequencing. Methods We examined synovial tissue samples from 28 patients: six patients with ReA and nine with UA, and a control group consisting of seven patients with rheumatoid arthritis and six with osteoarthritis (OA). Using broad-range bacterial PCR producing a 1,400-base-pair fragment from the 16S rRNA gene, at least 24 clones were sequenced for each synovial tissue sample. To identify the corresponding bacteria, DNA sequences were compared with sequences from the EMBL (European Molecular Biology Laboratory) database. Results Bacterial DNA was detected in 75% of the 28 synovial tissue samples. DNA from 68 various bacterial species were found in ReA and UA samples, whereas DNA from 12 bacteria were detected in control group samples. Most of the bacterial DNAs detected were from skin or intestinal bacteria. DNA from bacteria known to trigger ReA, such as Shigella flexneri and Shigella sonnei, were detected in ReA and UA samples of synovial tissue and not in control samples. DNA from various bacterial species detected in this study have not previously been found in synovial samples. Conclusion This study is the first to use broad-range PCR targeting the full 16S rRNA gene for detection of bacterial DNA in synovial tissue. We detected DNA from a wide spectrum of bacterial species, including those known to be involved in ReA and others not previously associated with ReA or related arthritis. The pathogenic

  15. Automated Broad-Range Molecular Detection of Bacteria in Clinical Samples

    PubMed Central

    Hoogewerf, Martine; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.

    2016-01-01

    Molecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are specific for each microbial species. As this is an open technique, clinicians do not need to decide in advance what to look for. We compared routine culture to IS-pro using 66 samples sent in for routine bacterial diagnostic testing. The samples were obtained from patients with infections in normally sterile sites (without a resident microbiota). The results were identical in 20 (30%) samples, IS-pro detected more bacterial species than culture in 31 (47%) samples, and five of the 10 culture-negative samples were positive with IS-pro. The case histories of the five patients from whom these culture-negative/IS-pro-positive samples were obtained suggest that the IS-pro findings are highly clinically relevant. Our findings indicate that an open molecular approach, such as IS-pro, may have a high added value for clinical practice. PMID:26763956

  16. Conjugal Transfer of Broad-Host-Range Plasmid pAMβ1 into Enteric Species of Lactic Acid Bacteria

    PubMed Central

    Vescovo, M.; Morelli, L.; Bottazzi, V.; Gasson, M. J.

    1983-01-01

    The broad-host-range plasmid pAMβ1, which codes for erythromycin and lincomycin resistance, was transferred by conjugation into Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus salivarius. A novel 17-megadalton plasmid molecule was detected in the transconjugants, confirming the introduction of pAMβ1 into each species. Images PMID:16346389

  17. Promoting Equitable Biology Lab Instruction by Engaging All Students in a Broad Range of Science Practices: An Exploratory Study

    ERIC Educational Resources Information Center

    Strimaitis, Anna M.; Southerland, Sherry A.; Sampson, Victor; Enderle, Patrick; Grooms, Jonathon

    2017-01-01

    This study examines what students enrolled in the honors and general sections of a high school biology course offered at the same school learn when they have an opportunity to participate in a broad or narrow range of science practices during their laboratory experiences. The results of our analysis suggest that the students enrolled in the…

  18. A Questionnaire for Screening a Broad Range of DSM-Defined Anxiety Disorder Symptoms in Clinically Referred Children and Adolescents

    ERIC Educational Resources Information Center

    Muris, Peter; Dreessen, Laura; Bogels, Susan; Weckx, Miryam; van Melick, Marion

    2004-01-01

    Objective: To examine the reliability and validity of the 66-item Screen for Child Anxiety Related Emotional Disorders-Revised (SCARED-R), a questionnaire for measuring a broad range of DSM-defined anxiety disorder symptoms, in a sample of clinically referred youths. Method: The SCARED-R was administered to children/adolescents and their parents…

  19. Extending the wavelength range in the Oclaro high-brightness broad area modules

    NASA Astrophysics Data System (ADS)

    Pawlik, Susanne; Guarino, Andrea; Sverdlov, Boris; Müller, Jürgen; Button, Christopher; Arlt, Sebastian; Jaeggi, Dominik; Lichtenstein, Norbert

    2010-02-01

    The demand for high power laser diode modules in the wavelength range between 793 nm and 1060 nm has been growing continuously over the last several years. Progress in eye-safe fiber lasers requires reliable pump power at 793 nm, modules at 808 nm are used for small size DPSSL applications and fiber-coupled laser sources at 830 nm are used in printing industry. However, power levels achieved in this wavelength range have remained lower than for the 9xx nm range. Here we report on approaches to increasing the reliable power in our latest generations of high power pump modules in the wavelength range between 793 nm and 1060 nm.

  20. Isolation and characterization of a bacteriophage with broad host range, displaying potential in preventing bovine diarrhoea.

    PubMed

    Anand, Taruna; Vaid, Rajesh K; Bera, Bidhan Ch; Barua, Sanjay; Riyesh, T; Virmani, N; Yadav, Neeraj; Malik, Praveen

    2015-10-01

    Phage therapy has been previously tried for treatment of diarrhoea in calves, pigs and lambs but those trials were conducted without any detailed information of used phages. Here, we report isolation of a broad-spectrum phage which showed bactericidal activity against 47.3 % of calf diarrhoeal isolates of Escherichia coli, in vitro. The isolated phage resembled the characteristics of Myoviridae family and showed ~97 % similarity with earlier reported bacteriophages of sub family-Tevenvirinae, genus-T4-like virus, based on nucleotide sequence of major head protein-gp23 gene. The phage exhibits the potential to be used as drug substitute tool against E. coli causing diarrhoea in cattle in farm environments.

  1. Characterization of an Escherichia coli aromatic hydroxylase with a broad substrate range.

    PubMed

    Prieto, M A; Perez-Aranda, A; Garcia, J L

    1993-04-01

    The hpaB gene encoding an aromatic hydroxylase of Escherichia coli ATCC 11105, a penicillin G acylase-producing strain, has been cloned and expressed in E. coli K-12. This gene was located near the pacA gene coding for penicillin G acylase. The hydroxylase has a molecular mass of 59,000 Da, uses NADH as a cosubstrate, and was tentatively classified as a 4-hydroxyphenylacetic acid hydroxylase, albeit it exhibited a rather broad substrate specificity acting on different monohydric and dihydric phenols. E. coli W, C, and B as well as Klebsiella pneumoniae M5a1 and Kluyvera citrophila ATCC 21285 (a penicillin G acylase-producing strain) but not E. coli K-12 contained sequences homologous to hpaB. Our results support the hypothesis that hpaB is a component of the 4-hydroxyphenylacetic acid degradative pathway of E. coli W.

  2. Characterization of an Escherichia coli aromatic hydroxylase with a broad substrate range.

    PubMed Central

    Prieto, M A; Perez-Aranda, A; Garcia, J L

    1993-01-01

    The hpaB gene encoding an aromatic hydroxylase of Escherichia coli ATCC 11105, a penicillin G acylase-producing strain, has been cloned and expressed in E. coli K-12. This gene was located near the pacA gene coding for penicillin G acylase. The hydroxylase has a molecular mass of 59,000 Da, uses NADH as a cosubstrate, and was tentatively classified as a 4-hydroxyphenylacetic acid hydroxylase, albeit it exhibited a rather broad substrate specificity acting on different monohydric and dihydric phenols. E. coli W, C, and B as well as Klebsiella pneumoniae M5a1 and Kluyvera citrophila ATCC 21285 (a penicillin G acylase-producing strain) but not E. coli K-12 contained sequences homologous to hpaB. Our results support the hypothesis that hpaB is a component of the 4-hydroxyphenylacetic acid degradative pathway of E. coli W. Images PMID:8458860

  3. Study of transmission line attenuation in broad band millimeter wave frequency range

    SciTech Connect

    Pandya, Hitesh Kumar B.; Austin, M. E.; Ellis, R. F.

    2013-10-15

    Broad band millimeter wave transmission lines are used in fusion plasma diagnostics such as electron cyclotron emission (ECE), electron cyclotron absorption, reflectometry and interferometry systems. In particular, the ECE diagnostic for ITER will require efficient transmission over an ultra wide band, 100 to 1000 GHz. A circular corrugated waveguide transmission line is a prospective candidate to transmit such wide band with low attenuation. To evaluate this system, experiments of transmission line attenuation were performed and compared with theoretical loss calculations. A millimeter wave Michelson interferometer and a liquid nitrogen black body source are used to perform all the experiments. Atmospheric water vapor lines and continuum absorption within this band are reported. Ohmic attenuation in corrugated waveguide is very low; however, there is Bragg scattering and higher order mode conversion that can cause significant attenuation in this transmission line. The attenuation due to miter bends, gaps, joints, and curvature are estimated. The measured attenuation of 15 m length with seven miter bends and eighteen joints is 1 dB at low frequency (300 GHz) and 10 dB at high frequency (900 GHz), respectively.

  4. Cobalamin riboswitches exhibit a broad range of ability to discriminate between methylcobalamin and adenosylcobalamin.

    PubMed

    Polaski, Jacob T; Webster, Samantha M; Johnson, James E; Batey, Robert T

    2017-07-14

    Riboswitches are a widely distributed class of regulatory RNAs in bacteria that modulate gene expression via small-molecule-induced conformational changes. Generally, these RNA elements are grouped into classes based upon conserved primary and secondary structure and their cognate effector molecule. Although this approach has been very successful in identifying new riboswitch families and defining their distributions, small sequence differences between structurally related RNAs can alter their ligand selectivity and regulatory behavior. Herein, we use a structure-based mutagenic approach to demonstrate that cobalamin riboswitches have a broad spectrum of preference for the two biological forms of cobalamin in vitro using isothermal titration calorimetry. This selectivity is primarily mediated by the interaction between a peripheral element of the RNA that forms a T-loop module and a subset of nucleotides in the cobalamin-binding pocket. Cell-based fluorescence reporter assays in Escherichia coli revealed that mutations that switch effector preference in vitro lead to differential regulatory responses in a biological context. These data demonstrate that a more comprehensive analysis of representative sequences of both previously and newly discovered classes of riboswitches might reveal subgroups of RNAs that respond to different effectors. Furthermore, this study demonstrates a second distinct means by which tertiary structural interactions in cobalamin riboswitches dictate ligand selectivity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Fluorographene Modified by Grignard Reagents: A Broad Range of Functional Nanomaterials.

    PubMed

    Mazánek, Vlastimil; Libánská, Alena; Šturala, Jiří; Bouša, Daniel; Sedmidubský, David; Pumera, Martin; Janoušek, Zbyněk; Plutnar, Jan; Sofer, Zdeněk

    2017-02-03

    Fluorographene is the youngest stoichiometric derivative of graphene; hence, its reactivity is only poorly explored. Compared to graphene, the significantly higher reactivity of C-F bonds makes this material a suitable platform for a large number of chemical modifications. Fluorographene is also the only member of the halographene family that can be prepared in the stoichiometric composition (C1 F1 ). Herein, the chemical modification of fluorographene with Grignard reagents, which are well known in organic synthesis for the formation of new C-C bonds, is presented. The reaction with alkyl magnesium bromides led to successful modification of fluorographene with ethyl, vinyl, ethynyl and propargyl groups. Chemical characterisation showed the presence of covalently bonded functional groups in a high concentration exceeding one functional group per C6 motif. The reactivity of Grignard reagents with fluorographene decreased from ethyl to ethynyl. The terminal carbon-carbon triple bonds were used for click reactions with organic azides leading to the formation of triazole rings. These findings open up a broad spectrum of opportunities for simple and robust modification of graphene by chemical reactions proceeding at room temperature under mild conditions. These results have major application potential in sensing, biomedical and energy-related applications.

  6. Determination of metal ions by fluorescence anisotropy exhibits a broad dynamic range

    NASA Astrophysics Data System (ADS)

    Thompson, Richard B.; Maliwal, Badri P.; Fierke, Carol A.

    1998-05-01

    Recently, we have shown that metal ions free in solution may be determined at low levels by fluorescence anisotropy (polarization) measurements. Anisotropy measurements enjoy the advantages of wavelength ratiometric techniques for determining metal ions such as calcium, because anisotropy measurements are ratiometric as well. Furthermore, fluorescence anisotropy may be imaged in the microscope. An advantage of anisotropy not demonstrated for wavelength ratiometric approaches using indicators such as Fura-2 and Indo-1 is that under favorable circumstances anisotropy-based determinations exhibit a much broader dynamic range in metal ion concentration. Determinations of free Zn(II) in the picomolar range are demonstrated.

  7. Design, calibration and application of broad-range optical nanosensors for determining intracellular pH.

    PubMed

    Søndergaard, Rikke V; Henriksen, Jonas R; Andresen, Thomas L

    2014-12-01

    Particle-based nanosensors offer a tool for determining the pH in the endosomal-lysosomal system of living cells. Measurements providing absolute values of pH have so far been restricted by the limited sensitivity range of nanosensors, calibration challenges and the complexity of image analysis. This protocol describes the design and application of a polyacrylamide-based nanosensor (∼60 nm) that covalently incorporates two pH-sensitive fluorophores, fluorescein (FS) and Oregon Green (OG), to broaden the sensitivity range of the sensor (pH 3.1-7.0), and uses the pH-insensitive fluorophore rhodamine as a reference fluorophore. The nanosensors are spontaneously taken up via endocytosis and directed to the lysosomes where dynamic changes in pH can be measured with live-cell confocal microscopy. The most important focus areas of the protocol are the choice of pH-sensitive fluorophores, the design of calibration buffers, the determination of the effective range and especially the description of how to critically evaluate results. The entire procedure typically takes 2-3 weeks.

  8. Genome Analysis of a Novel Broad Host Range Proteobacteria Phage Isolated from a Bioreactor Treating Industrial Wastewater

    PubMed Central

    de Leeuw, Marina; Baron, Maayan; Brenner, Asher; Kushmaro, Ariel

    2017-01-01

    Bacteriophages are viruses that infect bacteria, and consequently they have a major impact on the development of a microbial population. In this study, the genome of a novel broad host range bacteriophage, Aquamicrobium phage P14, isolated from a wastewater treatment plant, was analyzed. The Aquamicrobium phage P14 was found to infect members of different Proteobacteria classes (Alphaproteobacteria and Betaproteobacteria). This phage contains a 40,551 bp long genome and 60% of its genes had blastx hits. Furthermore, the bacteriophage was found to share more than 50% of its genes with several podoviruses and has the same gene order as other polyvalent bacteriophages. The results obtained in this study led to the conclusion that indeed general features of the genome of the Aquamicrobium phage P14 are shared with other broad host range bacteriophages, however further analysis of the genome is needed in order to identify the specific mechanisms which enable the bacteriophage to infect both Alphaproteobacteria and Betaproteobacteria. PMID:28106814

  9. Relaxor ferroelectric-based electrocaloric polymer nanocomposites with a broad operating temperature range and high cooling energy.

    PubMed

    Li, Qi; Zhang, Guangzu; Zhang, Xiaoshan; Jiang, Shenglin; Zeng, Yike; Wang, Qing

    2015-04-01

    Electrocaloric nanocomposites simultaneously derive high electrocaloric strength from inorganic inclusions and high dielectric strength from the polymer matrix to display a pronounced electrocaloric effect (ECE). By designing the inorganic filler and polymer matrix, which are both relaxor ferroelectrics with the ambient-temperature phase transition and minimized hysteresis, a large ECE becomes accessible with high cooling efficiency over a broad temperature range at and near room temperature.

  10. Broad-range neutron spectra identification in ultraintense laser interactions with carbon-deuterated plasma

    SciTech Connect

    Youssef, A.; Kodama, R.; Habara, H.; Tanaka, K.A.; Sentoku, Y.; Tampo, M.; Toyama, Y.

    2005-11-15

    Detailed neutron energy spectra produced from a CD2 target irradiated by a 450 fs, 20 J, 1053 nm laser at an intensity of 3x10{sup 18} W/cm{sup 2} have been studied. Wide-ranging neutron spectra were observed from two different observation angles 20 deg. and 70 deg. relative to the rear-side target normal. The experiment and numerically calculated spectra, by a three-dimensional Monte Carlo code, indicate that the range of the measured spectra is larger than that produced by the D(d,n){sup 3}He reaction. An interpretation for the measured spectra is introduced by considering the {sup 12}C(d,n){sup 13}N and D({sup 12}c,n){sup 13}N reactions. In addition, the study revealed that the neutron spectra produced by the D-C and C-D reactions can overlap that produced by the D-D reaction, and due to their high cross sections, comparing to the D-D reaction, both of them effectively participate in the neutron yield.

  11. Open-sandwich immunoassay for sensitive and broad-range detection of a shellfish toxin gonyautoxin.

    PubMed

    Hara, Yuko; Dong, Jinhua; Ueda, Hiroshi

    2013-09-02

    At present, the analytical method for paralytic shellfish poisoning (PSP) toxins in shellfish is the mouse bioassay (MBA), which is an official method of the Association of Analytical Communities (AOAC [8]). However, the low sensitivity and concerns over the number of live animals required for testing have been cited as the major reason for seeking its replacement. In this report, we employed an open-sandwich immunoassay (OS-IA) to detect gonyautoxin (GTX2/3), a kind of PSP toxins. OS-IA, which utilizes the antigen-induced enhancement of antibody VH/VL interaction, can measure a small molecule antigen in a noncompetitive format. Hence it has a wider working range and shorter measurement time. We isolated anti-GTX2/3 antibody gene from a hybridoma GT-13A by screening a Fab-displaying phage library. Then the vectors for OS-IA were constructed, and examined for antigen concentration-dependency of the VH/VL interaction by OS-ELISA. As a result, in each case, signal intensity increases notably in a wide concentration range (0.1 to >1000 ng mL(-1)) of free GTX2/3, which was enough to cover its regulation value (80 μg 100 g(-1)) in many countries. So OS-IA will be widely applicable to detect PSP toxins in shellfish meats and in drinking water.

  12. Discovery of parvovirus-related sequences in an unexpected broad range of animals

    PubMed Central

    François, S.; Filloux, D.; Roumagnac, P.; Bigot, D.; Gayral, P.; Martin, D. P.; Froissart, R.; Ogliastro, M.

    2016-01-01

    Our knowledge of the genetic diversity and host ranges of viruses is fragmentary. This is particularly true for the Parvoviridae family. Genetic diversity studies of single stranded DNA viruses within this family have been largely focused on arthropod- and vertebrate-infecting species that cause diseases of humans and our domesticated animals: a focus that has biased our perception of parvovirus diversity. While metagenomics approaches could help rectify this bias, so too could transcriptomics studies. Large amounts of transcriptomic data are available for a diverse array of animal species and whenever this data has inadvertently been gathered from virus-infected individuals, it could contain detectable viral transcripts. We therefore performed a systematic search for parvovirus-related sequences (PRSs) within publicly available transcript, genome and protein databases and eleven new transcriptome datasets. This revealed 463 PRSs in the transcript databases of 118 animals. At least 41 of these PRSs are likely integrated within animal genomes in that they were also found within genomic sequence databases. Besides illuminating the ubiquity of parvoviruses, the number of parvoviral sequences discovered within public databases revealed numerous previously unknown parvovirus-host combinations; particularly in invertebrates. Our findings suggest that the host-ranges of extant parvoviruses might span the entire animal kingdom. PMID:27600734

  13. Near-linear response of mean monsoon strength to a broad range of radiative forcings.

    PubMed

    Boos, William R; Storelvmo, Trude

    2016-02-09

    Theoretical models have been used to argue that seasonal mean monsoons will shift abruptly and discontinuously from wet to dry stable states as their radiative forcings pass a critical threshold, sometimes referred to as a "tipping point." Further support for a strongly nonlinear response of monsoons to radiative forcings is found in the seasonal onset of the South Asian summer monsoon, which is abrupt compared with the annual cycle of insolation. Here it is shown that the seasonal mean strength of monsoons instead exhibits a nearly linear dependence on a wide range of radiative forcings. First, a previous theory that predicted a discontinuous, threshold response is shown to omit a dominant stabilizing term in the equations of motion; a corrected theory predicts a continuous and nearly linear response of seasonal mean monsoon strength to forcings. A comprehensive global climate model is then used to show that the seasonal mean South Asian monsoon exhibits a near-linear dependence on a wide range of isolated greenhouse gas, aerosol, and surface albedo forcings. This model reproduces the observed abrupt seasonal onset of the South Asian monsoon but produces a near-linear response of the mean monsoon by changing the duration of the summer circulation and the latitude of that circulation's ascent branch. Thus, neither a physically correct theoretical model nor a comprehensive climate model support the idea that seasonal mean monsoons will undergo abrupt, nonlinear shifts in response to changes in greenhouse gas concentrations, aerosol emissions, or land surface albedo.

  14. Differential effects of melatonin as a broad range UV-damage preventive dermato-endocrine regulator.

    PubMed

    Kleszczyński, Konrad; Hardkop, Lena H; Fischer, Tobias W

    2011-01-01

    Melatonin or N-acetyl-5-methoxytryptamine, is a compound derived from tryptophan that is found in all organisms from single cells to vertebrates and the human. It is one of the most evolutionarily conserved and pleiotropic hormone still active in humans and has been implicated in vital skin functions such as hair growth, fur pigmentation as well as melanoma control. Being a main secretory product of the pineal gland, melatonin regulates seasonal biorhythms, reproductive mechanisms or mammary gland metabolism. Due to its wide range endocrine properties it is also recognized to modulate numerous additional functions ranging from scavenging free radicals, immunomodulation-mediated DNA repair, wound healing, involvement in gene expression connected with circadian clocks and modulation of secondary endocrine signaling including prolactin release. Recently, apart from above mentioned entities, it was shown that melatonin suppresses ultraviolet (UV)-induced damage in human skin and human derived cell lines (e.g., keratinocytes, fibroblasts). The magnitude of UV-induced damage is mediated apparently by various molecular mechanisms related to generation of reactive oxygen species (ROS), apoptosis and mitochondrial-mediated cell death which are all counteracted or modulated by melatonin. We provide here an update of the relevant protective effects and molecular mechanisms of action of melatonin in the skin.

  15. Experimental study of attenuation and dispersion over a broad frequency range: 1. The apparatus

    NASA Astrophysics Data System (ADS)

    Takei, Yasuko; Fujisawa, Kazuhiro; McCarthy, Christine

    2011-09-01

    We have developed a new experimental method to investigate the anelasticity of a polycrystalline solid at high homologous temperature (T/Tm = 0.6-0.7). By using a rock analogue (borneol polycrystals) and a partially molten rock analogue (borneol + melt) having low melting and eutectic temperatures (Tm = 204.5°C, Te = 43°C), respectively, testing high homologous temperature can occur near room temperature. An apparatus is described for cyclic compressive, Young's modulus type attenuation experiments over a wide range of frequencies (10-10-4 Hz) and at small strain amplitudes (10-6-10-5). Creep tests to measure steady state viscosity can also be performed. Details of the apparatus, including its piezoelectric actuator, dual laser displacement meters and trio of load cells, are discussed. Attenuation spectra and modulus dispersion measured for aluminum, acrylic plastic, and a rock analogue are presented to illustrate the accuracy of the method.

  16. Future water demand in California under a broad range of land use scenarios

    NASA Astrophysics Data System (ADS)

    Wilson, T. S.; Sleeter, B. M.; Cameron, D. R.

    2016-12-01

    California continues to be gripped by the most severe drought on record. Most general circulation models agree the state will continue to warm this century and research suggests persistent, long-term droughts may become the new normal, exacerbating an already uncertain water supply future. Population increases and agricultural intensification will likely stress existing, highly variable inter-annual water supplies even further in coming decades. Using the Land Use and Carbon Scenario Simulator (LUCAS) model, we explore a wide range of potential water demand futures from 2012 to 2062 based on 8 alternative, spatially-explicit (1 km) land use scenarios and land-use related water demand. Scenarios include low and high rates for urbanization, agricultural expansion, and agricultural contraction as well as lowest and highest rates for the combined suite of anthropogenic land uses. Land change values were sampled from county-level historical (1991-2012) land change data and county-level average water use data for urban areas (i.e. municipal and industrial) and annual and perennial cropland. We modeled 100 Monte Carlo simulations for each scenario to better characterize and capture model uncertainty and a range of potential future outcomes. Results show water demand in Mediterranean California was lowest in the low anthropogenic change scenario, dropping an average 2.7 million acre feet (MAF) by 2062. The highest water demand was seen in the high urbanization (+3.2 MAF), high agricultural expansion (+4.1 MAF), and the high anthropogenic (+4.3 MAF) scenarios. Results provide water managers and policy makers with information on diverging land use and water use futures, based on observed land change and water use trends, helping better inform land and resource management decisions.

  17. A broad pH range and processive chitinase from a metagenome library

    PubMed Central

    Thimoteo, S.S.; Glogauer, A.; Faoro, H.; de Souza, E.M.; Huergo, L.F.; Moerschbacher, B.M.; Pedrosa, F.O.

    2017-01-01

    Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin. PMID:28076454

  18. Near-linear response of mean monsoon strength to a broad range of radiative forcings

    PubMed Central

    Boos, William R.; Storelvmo, Trude

    2016-01-01

    Theoretical models have been used to argue that seasonal mean monsoons will shift abruptly and discontinuously from wet to dry stable states as their radiative forcings pass a critical threshold, sometimes referred to as a “tipping point.” Further support for a strongly nonlinear response of monsoons to radiative forcings is found in the seasonal onset of the South Asian summer monsoon, which is abrupt compared with the annual cycle of insolation. Here it is shown that the seasonal mean strength of monsoons instead exhibits a nearly linear dependence on a wide range of radiative forcings. First, a previous theory that predicted a discontinuous, threshold response is shown to omit a dominant stabilizing term in the equations of motion; a corrected theory predicts a continuous and nearly linear response of seasonal mean monsoon strength to forcings. A comprehensive global climate model is then used to show that the seasonal mean South Asian monsoon exhibits a near-linear dependence on a wide range of isolated greenhouse gas, aerosol, and surface albedo forcings. This model reproduces the observed abrupt seasonal onset of the South Asian monsoon but produces a near-linear response of the mean monsoon by changing the duration of the summer circulation and the latitude of that circulation’s ascent branch. Thus, neither a physically correct theoretical model nor a comprehensive climate model support the idea that seasonal mean monsoons will undergo abrupt, nonlinear shifts in response to changes in greenhouse gas concentrations, aerosol emissions, or land surface albedo. PMID:26811462

  19. A broad pH range and processive chitinase from a metagenome library.

    PubMed

    Thimoteo, S S; Glogauer, A; Faoro, H; de Souza, E M; Huergo, L F; Moerschbacher, B M; Pedrosa, F O

    2017-01-05

    Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N'-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

  20. In situ Raman probing of graphene over a broad doping range upon rubidium vapor exposure.

    PubMed

    Parret, Romain; Paillet, Matthieu; Huntzinger, Jean-Roch; Nakabayashi, Denise; Michel, Thierry; Tiberj, Antoine; Sauvajol, Jean-Louis; Zahab, Ahmed A

    2013-01-22

    We report in situ Raman scattering experiments on single-layer graphene (SLG) and Bernal bilayer graphene (BLG) during exposure to rubidium vapor. The G- and 2D-band evolutions with doping time are presented and analyzed. On SLG, the extended doping range scanned (up to about 10(14) electrons/cm(2)) allows the observation of three regimes in the evolution of the G-band frequency: a continuous upshift followed by a plateau and a downshift. Overall the measured evolution is interpreted as the signature of the competition between dynamic and adiabatic effects upon n-doping. Comparison of the obtained results with theoretical predictions indicates however that a substrate pinning effect occurs and inhibits charge-induced lattice expansion of SLG. At low doping, a direct link between electrostatic gating and Rb doping results is presented. For BLG, the added electrons are shown to be first confined in the top layer, but the system evolves with time toward a more symmetric repartition of the added electrons in both layers. The results obtained on BLG also confirm that the slope of the phonon dispersion close to the K point tends to be slightly reduced at low doping but suggest the occurrence of an unexpected increase of the phonon dispersion slope at higher electron concentration.

  1. Numerical Upscaling of Transport Through Obstructed Regions Over a Broad Range of Reynolds Numbers

    NASA Astrophysics Data System (ADS)

    Sund, N. L.; Bolster, D.; Mattis, S. A.

    2014-12-01

    While historically flow and transport in porous media has focused on low Reynolds number and Peclet number regimes there are a variety of examples relevant to environmental fluid dynamics where higher Reynolds number flows are important. A common example might include flow and transport through wetlands where plants act as the solid phase of an effective porous medium. In particular, heterogeneity in the flow field due to presence of the solid phases gives rise to complex transport and mixing behaviors that cannot be upscaled at pre-asymptotic times using conventional approaches. We numerically simulate pore-scale flow and transport through obstructed domains over a range of Reynolds numbers from 15 to 280 and then upscale transport. We upscale using a correlated continuous time random walk (correlated CTRW) model, originally introduced in [1]. We then assess the correlated CTRW's ability to predict observables for both asymptotic and pre-asymptotic time scales and compare our results to those of a classical CTRW to determine when velocity correlations must be accounted for. REFERENCES[1] T.L. Borgne, M. Dentz, J. Carrera: Lagrangian statistical model for transport in highly heterogeneous velocity fields, Physical Review Letters 101 (2008) 090601.

  2. A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria

    PubMed Central

    Monson, Rita; Smith, Debra S.; Matilla, Miguel A.; Roberts, Kevin; Richardson, Elizabeth; Drew, Alison; Williamson, Neil; Ramsay, Josh; Welch, Martin; Salmond, George P. C.

    2015-01-01

    Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways. PMID:26733980

  3. Investigations of Saturn’s Main Rings over Broad Range of Wavelengths

    NASA Astrophysics Data System (ADS)

    Spilker, Linda J.; Deau, Estelle; Morishima, Ryuji; Filacchione, Gianrico; Hedman, Matt; Nicholson, Phil; Colwell, Josh; Bradley, Todd; Showalter, Mark; Pilorz, Stu; Brooks, Shawn; Ciarniello, Mauro

    2015-11-01

    An abundance of information about the characteristics of Saturn’s ring particles and their regolith can be obtained by comparing the changes in their brightness, color and temperature with changing viewing geometry over a wide range of wavelengths from ultraviolet through the thermal infrared. Data from Cassini’s Composite Infrared Spectrometer (CIRS), Visual and Infrared Mapping Spectrometer (VIMS), Imaging Science Subsystem (ISS) and Ultraviolet Imaging Spectrograph (UVIS) are jointly studied using data from the lit and unlit main rings at multiple geometries and solar elevations over 11 years of the Cassini mission. Using multi-wavelength data sets allows us to test different thermal models by combining the effects of particle albedo, regolith grain size and surface roughness with thermal emissivity and inertia, particle spin rate and spin axis orientation.CIRS temperatures, ISS colors and UVIS brightness appear to vary noticeably with phase angle, but are not a strong function of spacecraft elevation angle. Color, temperature and brightness dependence on solar elevation angle are also observed. VIMS observations show that the infrared ice absorption band depths change with the solar phase angle, in particular between 0-20° and at high phase. This trend indicates that single scattering approximation is correct only at low phases (<20°) while at high phase multiple scattering must be taken into account.These results imply that the individual properties of the ring particles may play a larger role than the collective properties of the rings, in particular at visible wavelengths. The temperature and color variation with phase angle may be a result of scattering within the regolith, as well as scattering between individual particles or clumps in a many-particle-thick layer. Initial results from our joint studies will be presented.This research was carried out in part at the Jet Propulsion Laboratory, California Institute of Technology, under contract with NASA

  4. A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria.

    PubMed

    Monson, Rita; Smith, Debra S; Matilla, Miguel A; Roberts, Kevin; Richardson, Elizabeth; Drew, Alison; Williamson, Neil; Ramsay, Josh; Welch, Martin; Salmond, George P C

    2015-01-01

    Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.

  5. Characterization of novel virulent broad-host-range phages of Xylella fastidiosa and Xanthomonas.

    PubMed

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

    2014-01-01

    The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ~4 × 10(-12) ml cell(-1) min(-1) for X. fastidiosa strain Temecula 1 and ~5 × 10(-10) to 7 × 10(-10) ml cell(-1) min(-1) for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa.

  6. Bi-Tapered Fiber Sensor Using a Supercontinuum Light Source for a Broad Spectral Range

    NASA Astrophysics Data System (ADS)

    Garcia Mina, Diego Felipe

    We describe the fabrication bi-tapered optical fiber sensors designed for shorter wavelength operation and we study their optical properties. The new sensing system designed and built for the project is a specialty optical fiber that is single-mode in the visible/near infrared wavelength region of interest. In fabricating the tapered fiber we control the taper parameters, such as the down-taper and up-taper rate, shape and length, and the fiber waist diameter and length. The sensing is mode is via the electromagnetic field, which is evanescent outside the optical fiber and is confined close to the fiber's surface (within a couple hundred nanometers). The fiber sensor system has multiple advantages as a compact, simple device with an ability to detected tiny changes in the refractive index. We developed a supercontinuum light source to provide a wide spectral wavelength range from visible to near IR. The source design was based on coupling light from a femtosecond laser in a photonic crystal fiber designed for high nonlinearity. The output light was efficiently coupled into the bi-tapered fiber sensor and good signal to noise was achieved across the wavelength region. The bi-tapered fiber starts and ends with a single mode fiber in the waist region there are many modes with different propagation constants that couple to the environment outside the fiber. The signals have a strong periodic component as the wavelength is scanned; we exploit the periodicity in the signal using a discrete Fourier transform analysis to correlate signal phase changes with the refractive index changes in the local environment. For small index changes we also measure a strong correlation with the dominant Fourier amplitude component. Our experiments show that our phase-based signal processing technique works well at shorter wavelengths and we extract a new feature, the Fourier amplitude, to measure the refractive index difference. We conducted experiments using aqueous medium with

  7. Kinetics of the NCN + NO reaction over a broad temperature and pressure range.

    PubMed

    Welz, Oliver; Olzmann, Matthias

    2012-07-12

    Rate coefficients for the reaction (3)NCN + NO → products (R3) were measured in the temperature range 251-487 K at pressures from 10 mbar up to 50 bar with helium as the bath gas. The experiments were carried out in slow-flow reactors by using pulsed excimer laser photolysis of NCN(3) at 193 or 248 nm for the production of NCN. Pseudo-first-order conditions ([NCN](0) ≪ NO) were applied, and NCN was detected time-resolved by resonant laser-induced fluorescence excited near 329 nm. The measurements at the highest pressures yielded values of k(3) ∼ 8 × 10(-12) cm(3) s(-1) virtually independent of temperature and pressure, which indicates a substantially smaller high-pressure limiting value of k(3) than predicted in earlier works. Our experiments at pressures below 1 bar confirm the negative temperature and positive pressure dependence of the rate coefficient k(3) found in previous investigations. The falloff behavior of k(3) was rationalized by a master equation analysis based on a barrierless association step (3)NCN + NO ↔ NCNNO((2)A″) followed by a fast internal conversion NCNNO((2)A″) ↔ NCNNO((2)A'). From 251-487 K and above 30 mbar, the rate coefficient k(3) is well represented by a Troe parametrization for a recombination/dissociation reaction, k(3)(T,P) = k(4)(∞)k(4)(0)[M]F(k(4)(0)[M] + k(4)(∞))(-1), where k(4) represents the rate coefficient for the recombination reaction (3)NCN + NO. The following parameters were determined (30% estimated error of the absolute value of k(3)): k(4)(0)[M=He] = 1.91 × 10(-30)(T/300 K)(-3.3) cm(6) s(-1)[He], k(4)(∞) = 1.12 × 10(-11) exp(-23 K/T) cm(3) s(-1), and F(C) = 0.28 exp(173 K/T).

  8. Multiple conformational states of the hammerhead ribozyme, broad time range of relaxation and topology of dynamics.

    PubMed

    Menger, M; Eckstein, F; Porschke, D

    2000-11-15

    The dynamics of a hammerhead ribozyme was analyzed by measurements of fluorescence-detected temperature jump relaxation. The ribozyme was substituted at different positions by 2-aminopurine (2-AP) as fluorescence indicator; these substitutions do not inhibit catalysis. The general shape of relaxation curves reported from different positions of the ribozyme is very similar: a fast decrease of fluorescence, mainly due to physical quenching, is followed by a slower increase of fluorescence due to conformational relaxation. In most cases at least three relaxation time constants in the time range from a few microseconds to approximately 200 ms are required for fitting. Although the relaxation at different positions of the ribozyme is similar in general, suggesting a global type of ribozyme dynamics, a close examination reveals differences, indicating an individual local response. For example, 2-AP in a tetraloop reports mainly the local loop dynamics known from isolated loops, whereas 2-AP located at the core, e.g. at the cleavage site or its vicinity, also reports relatively large amplitudes of slower components of the ribozyme dynamics. A variant with an A-->G substitution in domain II, resulting in an inactive form, leads to the appearance of a particularly slow relaxation process (tau approximately 200 ms). Addition of Mg(2+) ions induces a reduction of amplitudes and in most cases a general increase of time constants. Differences between the hammerhead variants are clearly demonstrated by subtraction of relaxation curves recorded under corresponding conditions. The changes induced in the relaxation response by Mg(2+) are very similar to those induced by Ca(2+). The relaxation data do not provide any evidence for formation of Mg(2+)-inner sphere complexes in hammerhead ribozymes, because a Mg(2+)-specific relaxation effect was not visible. However, a Mg(2+)-specific effect was found for a dodeca-riboadenylate substituted with 2-AP, showing that the fluorescence of 2

  9. Multiple conformational states of the hammerhead ribozyme, broad time range of relaxation and topology of dynamics

    PubMed Central

    Menger, Marcus; Eckstein, Fritz; Porschke, Dietmar

    2000-01-01

    The dynamics of a hammerhead ribozyme was analyzed by measurements of fluorescence-detected temperature jump relaxation. The ribozyme was substituted at different positions by 2-aminopurine (2-AP) as fluorescence indicator; these substitutions do not inhibit catalysis. The general shape of relaxation curves reported from different positions of the ribozyme is very similar: a fast decrease of fluorescence, mainly due to physical quenching, is followed by a slower increase of fluorescence due to conformational relaxation. In most cases at least three relaxation time constants in the time range from a few microseconds to ~200 ms are required for fitting. Although the relaxation at different positions of the ribozyme is similar in general, suggesting a global type of ribozyme dynamics, a close examination reveals differences, indicating an individual local response. For example, 2-AP in a tetraloop reports mainly the local loop dynamics known from isolated loops, whereas 2-AP located at the core, e.g. at the cleavage site or its vicinity, also reports relatively large amplitudes of slower components of the ribozyme dynamics. A variant with an A→G substitution in domain II, resulting in an inactive form, leads to the appearance of a particularly slow relaxation process (τ ≈200 ms). Addition of Mg2+ ions induces a reduction of amplitudes and in most cases a general increase of time constants. Differences between the hammerhead variants are clearly demonstrated by subtraction of relaxation curves recorded under corresponding conditions. The changes induced in the relaxation response by Mg2+ are very similar to those induced by Ca2+. The relaxation data do not provide any evidence for formation of Mg2+-inner sphere complexes in hammerhead ribozymes, because a Mg2+-specific relaxation effect was not visible. However, a Mg2+-specific effect was found for a dodeca-riboadenylate substituted with 2-AP, showing that the fluorescence of 2-AP is able to indicate inner sphere

  10. Influences of the common FTO rs9939609 variant on inflammatory markers throughout a broad range of body mass index.

    PubMed

    Zimmermann, Esther; Skogstrand, Kristin; Hougaard, David M; Astrup, Arne; Hansen, Torben; Pedersen, Oluf; Sørensen, Thorkild I A; Jess, Tine

    2011-01-05

    A recent study reported that the fatness associated A-allele of FTO rs9939609 increased plasma high sensitivity C-reactive protein (hs-CRP) levels independent of fatness. We aimed to investigate if this gene variant had fatness-independent effects on plasma hs-CRP and 10 additional circulating obesity-related adipokines throughout a broad range of body mass index (BMI) among Danish men. In a population of 362,200 young men, examined for military service between 1943 and 1977, two groups were identified: 1) a random 1% sample and 2) all obese men (BMI = 31.0 kg/m(2), all of whom were above the 99(th) percentile of this population). At an average age of 49 years (range: 39 through 65 years), 551 men, hereof 231 of the obese, were re-examined, including genotyping and measurement of the fasting circulating inflammatory markers hs-CRP, IL-1β, IL-6, IL-10, IL-18, mip1α, mip1β, sTNFα-R1, TGF-β, TNF-α and leptin. Men with known disease were excluded from the examination. All the inflammatory markers were log-transformed to approximate a normal distribution. Genotype-phenotype relationships were studied using linear regression analyses with the inflammatory markers as the response variable. Significant positive associations between hs-CRP, leptin and a broad range of BMI were observed, but the associations did not significantly differ across FTO rs9939609 genotype. There were no significant associations between the other inflammatory markers, FTO rs9939609 genotype or BMI, respectively. No fatness-independent effects of the FTO rs9939609 A-allele on a series of inflammatory markers were observed in this cohort of healthy middle-aged men representing a broad range of fatness.

  11. Influences of the Common FTO rs9939609 Variant on Inflammatory Markers Throughout a Broad Range of Body Mass Index

    PubMed Central

    Zimmermann, Esther; Skogstrand, Kristin; Hougaard, David M.; Astrup, Arne; Hansen, Torben; Pedersen, Oluf; Sørensen, Thorkild I. A.; Jess, Tine

    2011-01-01

    Background A recent study reported that the fatness associated A-allele of FTO rs9939609 increased plasma high sensitivity C-reactive protein (hs-CRP) levels independent of fatness. We aimed to investigate if this gene variant had fatness-independent effects on plasma hs-CRP and 10 additional circulating obesity-related adipokines throughout a broad range of body mass index (BMI) among Danish men. Methodology/Principal Findings In a population of 362,200 young men, examined for military service between 1943 and 1977, two groups were identified: 1) a random 1% sample and 2) all obese men (BMI = 31.0 kg/m2, all of whom were above the 99th percentile of this population). At an average age of 49 years (range: 39 through 65 years), 551 men, hereof 231 of the obese, were re-examined, including genotyping and measurement of the fasting circulating inflammatory markers hs-CRP, IL-1β, IL-6, IL-10, IL-18, mip1α, mip1β, sTNFα-R1, TGF-β, TNF-α and leptin. Men with known disease were excluded from the examination. All the inflammatory markers were log-transformed to approximate a normal distribution. Genotype-phenotype relationships were studied using linear regression analyses with the inflammatory markers as the response variable. Significant positive associations between hs-CRP, leptin and a broad range of BMI were observed, but the associations did not significantly differ across FTO rs9939609 genotype. There were no significant associations between the other inflammatory markers, FTO rs9939609 genotype or BMI, respectively. Conclusion No fatness-independent effects of the FTO rs9939609 A-allele on a series of inflammatory markers were observed in this cohort of healthy middle-aged men representing a broad range of fatness. PMID:21246032

  12. Miniature long-range light beam transmitter resorting to a high-power broad area laser diode

    NASA Astrophysics Data System (ADS)

    Yue, Wenjing; Lee, Sang-Shin

    2014-08-01

    A miniature long-range light beam transmitter, which taps into a high-power broad area laser diode (BALD), was realized to exhibit a uniform detectable width. An effective model was proposed to practically emulate the multimode characteristics of the beam generated by the BALD. The model, solely based on the emitting region and far-field divergence angle pertaining to the LD, is established through an incoherent superposition of multiple normalized Hermit-Gaussian modes. The feasibility of the proposed model was successfully verified in terms of the calculated and observed irradiance distributions of the light beams. A long-range light beam transmitter was then designed and constructed taking advantage of the BALD source in conjunction with a beam shaper. The manufactured transmitter was corroborated to provide an infrared beam with a constant detectable width of ~1 m, over a distance ranging up to 400 m, for a predefined threshold level.

  13. A broad range assay for rapid detection and etiologic characterization of bacterial meningitis: performance testing in samples from sub-Sahara☆, ☆☆,★

    PubMed Central

    Won, Helen; Yang, Samuel; Gaydos, Charlotte; Hardick, Justin; Ramachandran, Padmini; Hsieh, Yu-Hsiang; Kecojevic, Alexander; Njanpop-Lafourcade, Berthe-Marie; Mueller, Judith E.; Tameklo, Tsidi Agbeko; Badziklou, Kossi; Gessner, Bradford D.; Rothman, Richard E.

    2012-01-01

    This study aimed to conduct a pilot evaluation of broad-based multiprobe polymerase chain reaction (PCR) in clinical cerebrospinal fluid (CSF) samples compared to local conventional PCR/culture methods used for bacterial meningitis surveillance. A previously described PCR consisting of initial broad-based detection of Eubacteriales by a universal probe, followed by Gram typing, and pathogen-specific probes was designed targeting variable regions of the 16S rRNA gene. The diagnostic performance of the 16S rRNA assay in “”127 CSF samples was evaluated in samples from patients from Togo, Africa, by comparison to conventional PCR/culture methods. Our probes detected Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. Uniprobe sensitivity and specificity versus conventional PCR were 100% and 54.6%, respectively. Sensitivity and specificity of uniprobe versus culture methods were 96.5% and 52.5%, respectively. Gram-typing probes correctly typed 98.8% (82/83) and pathogen-specific probes identified 96.4% (80/83) of the positives. This broad-based PCR algorithm successfully detected and provided species level information for multiple bacterial meningitis agents in clinical samples. PMID:22809694

  14. Broad-Host-Range Plasmids from Agricultural Soils Have IncP-1 Backbones with Diverse Accessory Genes▿

    PubMed Central

    Sen, Diya; Van der Auwera, Geraldine A.; Rogers, Linda M.; Thomas, Christopher M.; Brown, Celeste J.; Top, Eva M.

    2011-01-01

    Broad-host-range plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to antibiotics and heavy metals or degradation of pollutants. Although some broad-host-range plasmids have been extensively studied, their evolutionary history and genetic diversity remain largely unknown. The goal of this study was to analyze and compare the genomes of 12 broad-host-range plasmids that were previously isolated from Norwegian soils by exogenous plasmid isolation and that encode mercury resistance. Complete nucleotide sequencing followed by phylogenetic analyses based on the relaxase gene traI showed that all the plasmids belong to one of two subgroups (β and ε) of the well-studied incompatibility group IncP-1. A diverse array of accessory genes was found to be involved in resistance to antimicrobials (streptomycin, spectinomycin, and sulfonamides), degradation of herbicides (2,4-dichlorophenoxyacetic acid and 2,4-dichlorophenoxypropionic acid), and a putative new catabolic pathway. Intramolecular transposition of insertion sequences followed by deletion was found to contribute to the diversity of some of these plasmids. The previous observation that the insertion sites of a Tn501-related element are identical in four IncP-1β plasmids (pJP4, pB10, R906, and R772) was further extended to three more IncP-1β plasmids (pAKD15, pAKD18, and pAKD29). We proposed a hypothesis for the evolution of these Tn501-bearing IncP-1β plasmids that predicts recent diversification followed by worldwide spread. Our study increases the available collection of complete IncP-1 plasmid genome sequences by 50% and will aid future studies to enhance our understanding of the evolution and function of this important plasmid family. PMID:21948829

  15. Intraspecific 16S rRNA gene diversity among clinical isolates of Neisseria species.

    PubMed

    Mechergui, Arij; Achour, Wafa; Hassen, Assia Ben

    2014-05-01

    In the present work, nearly the entire 16S rRNA gene sequences of 46 clinical samples of Neisseria spp. were determined, and the aligned sequences were analyzed to investigate the diversity of 16S rRNA genes in each commensal Neisseria species. Two 16S rRNA types were identified in two Neisseria sicca strains, three 16S rRNA types in five Neisseria macacae strains, fourteen 16S rRNA types in twenty Neisseria flavescens isolates, and fourteen 16S rRNA types in nineteen Neisseria mucosa isolates. The number of nucleotides that were different between 16S rRNA sequences within specie ranged from 1 to 15. We found high intraspecific sequence variation in 16S rRNA genes of Neisseria spp. strains. © 2013 APMIS. Published by John Wiley & Sons Ltd.

  16. Broad-range self-sweeping of a narrow-line self-pulsing Yb-doped fiber laser

    NASA Astrophysics Data System (ADS)

    Lobach, Ivan A.; Kablukov, Sergey I.; Podivilov, Evgeniy V.; Babin, Sergey A.

    2011-08-01

    The effect of broad-range (16 nm) self-sweeping of a narrow-line (less than 1 pm) Yb-doped fiber laser has been demonstrated experimentally. It is found that the effect arises from the self-sustained relaxation oscillations. As a result, the sweeping rate increases as square root of the laser power and decreases with increasing cavity length. Based on these results we propose a model describing dynamics of the laser frequency. The model takes into account the effects of gain saturation at the laser transition and spatial hole burning in the self-pulsing regime.

  17. Broad-range self-sweeping of a narrow-line self-pulsing Yb-doped fiber laser.

    PubMed

    Lobach, Ivan A; Kablukov, Sergey I; Podivilov, Evgeniy V; Babin, Sergey A

    2011-08-29

    The effect of broad-range (16 nm) self-sweeping of a narrow-line (less than 1 pm) Yb-doped fiber laser has been demonstrated experimentally. It is found that the effect arises from the self-sustained relaxation oscillations. As a result, the sweeping rate increases as square root of the laser power and decreases with increasing cavity length. Based on these results we propose a model describing dynamics of the laser frequency. The model takes into account the effects of gain saturation at the laser transition and spatial hole burning in the self-pulsing regime.

  18. Antiviral Activity of Favipiravir (T-705) against a Broad Range of Paramyxoviruses In Vitro and against Human Metapneumovirus in Hamsters

    PubMed Central

    Jochmans, D.; van Nieuwkoop, S.; Smits, S. L.; Neyts, J.; Fouchier, R. A. M.

    2016-01-01

    The clinical impact of infections with respiratory viruses belonging to the family Paramyxoviridae argues for the development of antiviral therapies with broad-spectrum activity. Favipiravir (T-705) has demonstrated potent antiviral activity against multiple RNA virus families and is presently in clinical evaluation for the treatment of influenza. Here we demonstrate in vitro activity of T-705 against the paramyxoviruses human metapneumovirus (HMPV), respiratory syncytial virus, human parainfluenza virus, measles virus, Newcastle disease virus, and avian metapneumovirus. In addition, we demonstrate activity against HMPV in hamsters. T-705 treatment inhibited replication of all paramyxoviruses tested in vitro, with 90% effective concentration (EC90) values of 8 to 40 μM. Treatment of HMPV-challenged hamsters with T-705 at 200 mg/kg of body weight/day resulted in 100% protection from infection of the lungs. In all treated and challenged animals, viral RNA remained detectable in the respiratory tract. The observation that T-705 treatment had a significant effect on infectious viral titers, with a limited effect on viral genome titers, is in agreement with its proposed mode of action of viral mutagenesis. However, next-generation sequencing of viral genomes isolated from treated and challenged hamsters did not reveal (hyper)mutation. Polymerase activity assays revealed a specific effect of T-705 on the activity of the HMPV polymerase. With the reported antiviral activity of T-705 against a broad range of RNA virus families, this small molecule is a promising broad-range antiviral drug candidate for limiting the viral burden of paramyxoviruses and for evaluation for treatment of infections with (re)emerging viruses, such as the henipaviruses. PMID:27185803

  19. Antiviral Activity of Favipiravir (T-705) against a Broad Range of Paramyxoviruses In Vitro and against Human Metapneumovirus in Hamsters.

    PubMed

    Jochmans, D; van Nieuwkoop, S; Smits, S L; Neyts, J; Fouchier, R A M; van den Hoogen, B G

    2016-08-01

    The clinical impact of infections with respiratory viruses belonging to the family Paramyxoviridae argues for the development of antiviral therapies with broad-spectrum activity. Favipiravir (T-705) has demonstrated potent antiviral activity against multiple RNA virus families and is presently in clinical evaluation for the treatment of influenza. Here we demonstrate in vitro activity of T-705 against the paramyxoviruses human metapneumovirus (HMPV), respiratory syncytial virus, human parainfluenza virus, measles virus, Newcastle disease virus, and avian metapneumovirus. In addition, we demonstrate activity against HMPV in hamsters. T-705 treatment inhibited replication of all paramyxoviruses tested in vitro, with 90% effective concentration (EC90) values of 8 to 40 μM. Treatment of HMPV-challenged hamsters with T-705 at 200 mg/kg of body weight/day resulted in 100% protection from infection of the lungs. In all treated and challenged animals, viral RNA remained detectable in the respiratory tract. The observation that T-705 treatment had a significant effect on infectious viral titers, with a limited effect on viral genome titers, is in agreement with its proposed mode of action of viral mutagenesis. However, next-generation sequencing of viral genomes isolated from treated and challenged hamsters did not reveal (hyper)mutation. Polymerase activity assays revealed a specific effect of T-705 on the activity of the HMPV polymerase. With the reported antiviral activity of T-705 against a broad range of RNA virus families, this small molecule is a promising broad-range antiviral drug candidate for limiting the viral burden of paramyxoviruses and for evaluation for treatment of infections with (re)emerging viruses, such as the henipaviruses.

  20. DegePrime, a Program for Degenerate Primer Design for Broad-Taxonomic-Range PCR in Microbial Ecology Studies

    PubMed Central

    Hugerth, Luisa W.; Wefer, Hugo A.; Lundin, Sverker; Jakobsson, Hedvig E.; Lindberg, Mathilda; Rodin, Sandra; Engstrand, Lars

    2014-01-01

    The taxonomic composition of a microbial community can be deduced by analyzing its rRNA gene content by, e.g., high-throughput DNA sequencing or DNA chips. Such methods typically are based on PCR amplification of rRNA gene sequences using broad-taxonomic-range PCR primers. In these analyses, the use of optimal primers is crucial for achieving an unbiased representation of community composition. Here, we present the computer program DegePrime that, for each position of a multiple sequence alignment, finds a degenerate oligomer of as high coverage as possible and outputs its coverage among taxonomic divisions. We show that our novel heuristic, which we call weighted randomized combination, performs better than previously described algorithms for solving the maximum coverage degenerate primer design problem. We previously used DegePrime to design a broad-taxonomic-range primer pair that targets the bacterial V3-V4 region (341F-805R) (D. P. Herlemann, M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek, and A. F. Andersson, ISME J. 5:1571–1579, 2011, http://dx.doi.org/10.1038/ismej.2011.41), and here we use the program to significantly increase the coverage of a primer pair (515F-806R) widely used for Illumina-based surveys of bacterial and archaeal diversity. By comparison with shotgun metagenomics, we show that the primers give an accurate representation of microbial diversity in natural samples. PMID:24928874

  1. DegePrime, a program for degenerate primer design for broad-taxonomic-range PCR in microbial ecology studies.

    PubMed

    Hugerth, Luisa W; Wefer, Hugo A; Lundin, Sverker; Jakobsson, Hedvig E; Lindberg, Mathilda; Rodin, Sandra; Engstrand, Lars; Andersson, Anders F

    2014-08-01

    The taxonomic composition of a microbial community can be deduced by analyzing its rRNA gene content by, e.g., high-throughput DNA sequencing or DNA chips. Such methods typically are based on PCR amplification of rRNA gene sequences using broad-taxonomic-range PCR primers. In these analyses, the use of optimal primers is crucial for achieving an unbiased representation of community composition. Here, we present the computer program DegePrime that, for each position of a multiple sequence alignment, finds a degenerate oligomer of as high coverage as possible and outputs its coverage among taxonomic divisions. We show that our novel heuristic, which we call weighted randomized combination, performs better than previously described algorithms for solving the maximum coverage degenerate primer design problem. We previously used DegePrime to design a broad-taxonomic-range primer pair that targets the bacterial V3-V4 region (341F-805R) (D. P. Herlemann, M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek, and A. F. Andersson, ISME J. 5:1571-1579, 2011, http://dx.doi.org/10.1038/ismej.2011.41), and here we use the program to significantly increase the coverage of a primer pair (515F-806R) widely used for Illumina-based surveys of bacterial and archaeal diversity. By comparison with shotgun metagenomics, we show that the primers give an accurate representation of microbial diversity in natural samples. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Emerging Trends in Molecular Interactions between Plants and the Broad Host Range Fungal Pathogens Botrytis cinerea and Sclerotinia sclerotiorum

    PubMed Central

    Mbengue, Malick; Navaud, Olivier; Peyraud, Rémi; Barascud, Marielle; Badet, Thomas; Vincent, Rémy; Barbacci, Adelin; Raffaele, Sylvain

    2016-01-01

    Fungal plant pathogens are major threats to food security worldwide. Sclerotinia sclerotiorum and Botrytis cinerea are closely related Ascomycete plant pathogens causing mold diseases on hundreds of plant species. There is no genetic source of complete plant resistance to these broad host range pathogens known to date. Instead, natural plant populations show a continuum of resistance levels controlled by multiple genes, a phenotype designated as quantitative disease resistance. Little is known about the molecular mechanisms controlling the interaction between plants and S. sclerotiorum and B. cinerea but significant advances were made on this topic in the last years. This minireview highlights a selection of nine themes that emerged in recent research reports on the molecular bases of plant-S. sclerotiorum and plant-B. cinerea interactions. On the fungal side, this includes progress on understanding the role of oxalic acid, on the study of fungal small secreted proteins. Next, we discuss the exchanges of small RNA between organisms and the control of cell death in plant and fungi during pathogenic interactions. Finally on the plant side, we highlight defense priming by mechanical signals, the characterization of plant Receptor-like proteins and the hormone abscisic acid in the response to B. cinerea and S. sclerotiorum, the role of plant general transcription machinery and plant small bioactive peptides. These represent nine trends we selected as remarkable in our understanding of fungal molecules causing disease and plant mechanisms associated with disease resistance to two devastating broad host range fungi. PMID:27066056

  3. Broad-spectral-range synchronized flat-top arrayed-waveguide grating applied in a 225-channel cascaded spectrometer.

    PubMed

    Akca, B I; Doerr, C R; Sengo, G; Wörhoff, K; Pollnau, M; de Ridder, R M

    2012-07-30

    We present a new synchronized design for flattening the passband of an arrayed-waveguide grating (AWG) over a broad wavelength range of 90 nm. A wavelength-insensitive 3-dB balanced coupler is designed to be used in duplicate in a Mach-Zehnder interferometer (MZI); the phase deviation created by one of the balanced couplers is cancelled by flipping the other coupler around. This MZI is arranged in tandem with the AWG such that the output signal of the MZI is the input signal of the AWG. We demonstrate a 5-channel, 18-nm-spacing AWG with a 0.5-dB bandwidth of 12 nm over a 90-nm spectral range. A low-loss cascaded AWG system is demonstrated by using the MZI-synchronized flat-top AWG as a primary filter.

  4. Single-ion polymer electrolyte membranes enable lithium-ion batteries with a broad operating temperature range.

    PubMed

    Cai, Weiwei; Zhang, Yunfeng; Li, Jing; Sun, Yubao; Cheng, Hansong

    2014-04-01

    Conductive processes involving lithium ions are analyzed in detail from a mechanistic perspective, and demonstrate that single ion polymeric electrolyte (SIPE) membranes can be used in lithium-ion batteries with a wide operating temperature range (25-80 °C) through systematic optimization of electrodes and electrode/electrolyte interfaces, in sharp contrast to other batteries equipped with SIPE membranes that display appreciable operability only at elevated temperatures (>60 °C). The performance is comparable to that of batteries using liquid electrolyte of inorganic salt, and the batteries exhibit excellent cycle life and rate performance. This significant widening of battery operation temperatures coupled with the inherent flexibility and robustness of the SIPE membranes makes it possible to develop thin and flexible Li-ion batteries for a broad range of applications.

  5. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome.

    PubMed

    Nossa, Carlos W; Oberdorf, William E; Yang, Liying; Aas, Jørn A; Paster, Bruce J; Desantis, Todd Z; Brodie, Eoin L; Malamud, Daniel; Poles, Michael A; Pei, Zhiheng

    2010-09-07

    To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome. A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species. Candidate primers evaluated were from the European rRNA database. To assess the effect of sequence length on accuracy of classification, 16S rRNA genes of various lengths were created by trimming the full length sequences. Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs. The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP). The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes, represented by 36 phyla. Truncation to 100 nucleotides (nt) downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87 (39.7%) of the 219 sequences, compared with misclassification of only 29 (13.2%) sequences with truncation to 350 nt. Among 350-nt sequence reads within various regions of the 16S rRNA gene, the reverse read of an amplicon generated using the 343F/798R primers had the least (8.2%) effect on classification. In comparison, truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0% of the 219 sequences. The 343F/798R amplicon accurately assigned 91.8% of the 219 sequences at the species level. Weighted by abundance of the species in the esophageal dataset, the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage (92%). Modification of the 343F/798R primers to 347F/803R increased their universality among foregut

  6. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

    PubMed Central

    Nossa, Carlos W; Oberdorf, William E; Yang, Liying; Aas, Jørn A; Paster, Bruce J; DeSantis, Todd Z; Brodie, Eoin L; Malamud, Daniel; Poles, Michael A; Pei, Zhiheng

    2010-01-01

    AIM: To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome. METHODS: A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species. Candidate primers evaluated were from the European rRNA database. To assess the effect of sequence length on accuracy of classification, 16S rRNA genes of various lengths were created by trimming the full length sequences. Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs. The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP). The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes, represented by 36 phyla. RESULTS: Truncation to 100 nucleotides (nt) downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87 (39.7%) of the 219 sequences, compared with misclassification of only 29 (13.2%) sequences with truncation to 350 nt. Among 350-nt sequence reads within various regions of the 16S rRNA gene, the reverse read of an amplicon generated using the 343F/798R primers had the least (8.2%) effect on classification. In comparison, truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0% of the 219 sequences. The 343F/798R amplicon accurately assigned 91.8% of the 219 sequences at the species level. Weighted by abundance of the species in the esophageal dataset, the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage (92%). Modification of the 343F/798R primers to 347F/803R increased their

  7. Dead-end hollow-fiber ultrafiltration for concentration and enumeration of Escherichia coli and broad-host-range plasmid DNA from wastewater

    PubMed Central

    Asfahl, Kyle L.; Savin, Mary C.

    2012-01-01

    Broad-host-range plasmids can facilitate dissemination of antibiotic resistance determinants among diverse bacterial populations. We evaluated hollow-fiber ultrafiltration for increases in detection efficiency of broad-host-range plasmids and Escherichia coli DNA in wastewater. Ultrafiltration followed by PCR showed limited increases in DNA detection and quantification in effluent compared with membrane filtration alone. PMID:22251424

  8. Population structure over a broad spatial scale driven by nonanthropogenic factors in a wide-ranging migratory mammal, Alaskan caribou.

    PubMed

    Mager, Karen H; Colson, Kevin E; Groves, Pam; Hundertmark, Kris J

    2014-12-01

    Wide-ranging mammals face significant conservation threats, and knowledge of the spatial scale of population structure and its drivers is needed to understand processes that maintain diversity in these species. We analysed DNA from 655 Alaskan caribou (Rangifer tarandus granti) from 20 herds that vary in population size, used 19 microsatellite loci to document genetic diversity and differentiation in Alaskan caribou, and examined the extent to which genetic differentiation was associated with hypothesized drivers of population subdivision including landscape features, population size and ecotype. We found that Alaskan caribou are subdivided into two hierarchically structured clusters: one group on the Alaska Peninsula containing discrete herds and one large group on the Mainland lacking differentiation between many herds. Population size, geographic distance, migratory ecotype and the Kvichak River at the nexus of the Alaska Peninsula were associated with genetic differentiation. Contrary to previous hypotheses, small Mainland herds were often differentiated genetically from large interconnected herds nearby, and genetic drift coupled with reduced gene flow may explain this pattern. Our results raise the possibility that behaviour helps to maintain genetic differentiation between some herds of different ecotypes. Alaskan caribou show remarkably high diversity and low differentiation over a broad geographic scale. These results increase information for the conservation of caribou and other migratory mammals threatened by population reductions and landscape barriers and may be broadly applicable to understanding the spatial scale and ecological drivers of population structure in widespread species. © 2014 John Wiley & Sons Ltd.

  9. Detection of fungal pathogens by a new broad range real-time PCR assay targeting the fungal ITS2 region.

    PubMed

    Zeller, Iris; Schabereiter-Gurtner, Claudia; Mihalits, Verena; Selitsch, Brigitte; Barousch, Wolfgang; Hirschl, Alexander M; Makristathis, Athanasios; Willinger, Birgit

    2017-09-08

    The rise in the incidence of fungal infections and the expanding spectrum of fungal pathogens make early and broad detection of fungal pathogens essential. In the present study, a panfungal real-time PCR assay for the broad-range detection of fungal DNA (Fungi assay) in a wide variety of clinical specimens was developed. Our in-house, HybProbe real-time PCR assay targets the ITS2 region of fungal DNA. The applicability was evaluated by testing 105 clinical samples from 98 patients with suspected fungal infection. Samples included tissue biopsies, paraffin embedded tissues, aspirates, EDTA-anticoagulated blood, cerebrospinal fluids and bronchoalveolar lavages. Fungal pathogens were identified by the Fungi assay in 47 samples. In all of these cases, conventional methods and clinical data were also indicative for a fungal infection. Five samples were interpreted false negative. blast analyses of the amplicons derived from 11 samples revealed the presence of environmental fungal species while other tests and clinical data did not suggest a fungal infection. This fact might indicate contaminated samples. The remaining 42 samples were negative by the Fungi assay as well as the conventional methods and were therefore regarded as true negatives. Thus, sensitivity was 90.4 % and specificity 79.2 %. The Fungi assay improved the targeted diagnosis of fungal infections allowing pathogen identification in samples that were histologically positive but culture negative. For reliable diagnosis, results have to be interpreted in context with conventional methods and clinical data.

  10. Comprehensive description of blood microbiome from healthy donors assessed by 16S targeted metagenomic sequencing.

    PubMed

    Païssé, Sandrine; Valle, Carine; Servant, Florence; Courtney, Michael; Burcelin, Rémy; Amar, Jacques; Lelouvier, Benjamin

    2016-05-01

    Recent studies have revealed that the blood of healthy humans is not as sterile as previously supposed. The objective of this study was to provide a comprehensive description of the microbiome present in different fractions of the blood of healthy individuals. The study was conducted in 30 healthy blood donors to the French national blood collection center (Établissement Français du Sang). We have set up a 16S rDNA quantitative polymerase chain reaction assay as well as a 16S targeted metagenomics sequencing pipeline specifically designed to analyze the blood microbiome, which we have used on whole blood as well as on different blood fractions (buffy coat [BC], red blood cells [RBCs], and plasma). Most of the blood bacterial DNA is located in the BC (93.74%), and RBCs contain more bacterial DNA (6.23%) than the plasma (0.03%). The distribution of 16S DNA is different for each fraction and spreads over a relatively broad range among donors. At the phylum level, blood fractions contain bacterial DNA mostly from the Proteobacteria phylum (more than 80%) but also from Actinobacteria, Firmicutes, and Bacteroidetes. At deeper taxonomic levels, there are striking differences between the bacterial profiles of the different blood fractions. We demonstrate that a diversified microbiome exists in healthy blood. This microbiome has most likely an important physiologic role and could be implicated in certain transfusion-transmitted bacterial infections. In this regard, the amount of 16S bacterial DNA or the microbiome profile could be monitored to improve the safety of the blood supply. © 2016 AABB.

  11. Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol

    SciTech Connect

    Allen, L.N.; Hanson, R.S.

    1985-03-01

    Four new cloning vectors have been constructed from the broad-host-range cloning vector pRK290. These vectors, pLA2901, pLA2905, pLA2910, and pLA2917, confer resistance to kanamycin and tetracycline. The latter two are cosmid derivatives of pLA2901. The new vectors can be mobilized into, and are stably maintained in, a variety of gram-negative bacteria. A Sau3A genomic bank of Methylobacterium organophilum strain xx DNA has been constructed in pLA2917, and complementation analysis, with a variety of mutants unable to grow on methanol, revealed at least five separate regions necessary for growth on methanol. Complementation analysis and Tn5 mutagenesis data suggest that at least three genes are responsible for expression of active methanol dehydrogenase.

  12. Stable isotope physiology of stem succulents across a broad range of volume-to-surface area ratio.

    PubMed

    Hultine, Kevin R; Williams, David G; Dettman, David L; Butterfield, Bradley J; Puente-Martinez, Raul

    2016-11-01

    Volume-to-surface area ratio (V:S) across stem succulent taxa varies by almost two orders of magnitude. The broad range in V:S of cacti and other succulent species likely has considerable importance for adaptation since stem volume determines the storage capacity of water, carbon and nutrients and stem surface area is directly related to whole-stem photosynthetic capacity. We examined the intrinsic physiological tradeoffs across diverse stem morphologies in three divergent evolutionary groups where stem succulence is common: Cactoideae, Opuntioideae (Cactaceae) and Euphorbiaceae. We predicted that variation in physiological response to environmental conditions would be (1) constrained by stem V:S, and (2) detectable in the stable isotope ratios of plant tissues. Stable isotope ratios were measured in the spines/prickles of 62 stem-succulent species occurring in a common garden setting in Phoenix, AZ, USA. Biomass δ(13)C, δ(2)H and δ(18)O increased with V:S in Cactoideae only, possibly reflecting various levels of Crassulacean acid metabolism (CAM) strength in the other lineages. Within Cactoideae-group with the highest CAM strength and largest range in V:S-δ(13)C and δ(18)O increased 2.2 and 11.5 ‰, respectively, with a 22-fold increase in V:S. Both δ(13)C and V:S decreased with species climate-niche estimates of precipitation, indicating that stem morphology and physiology in Cactoideae may be constrained by available moisture. Taken together, these data suggest that physiological tradeoffs associated with stem V:S are detectable across broad evolutionary groups despite differences in CAM strength.

  13. Observations of Heavy Element Abundances over a Broad Energy Range in 3He-rich Solar Energetic Particle Events

    NASA Astrophysics Data System (ADS)

    Wiedenbeck, M. E.; Mason, G. M.; Cohen, C. M.; Leske, R. A.; Cummings, A. C.; Dwyer, J. R.; Mazur, J. E.; Mewaldt, R. A.; Stone, E. C.; von Rosenvinge, T. T.

    2006-05-01

    During the maximum of solar cycle 23 a number of 3He-rich solar energetic particle (SEP) events with measurable intensities of heavy elements (Z≥6) at energies >10 MeV/nuc were observed with instrumentation on the Advanced Composition Explorer (ACE) spacecraft. This represents a relatively small fraction of all the 3He-rich SEP events that were detected since heavy-ion intensities at these energies were frequently too low to be measured. Using data from two ACE instruments (SIS covering ~10--60 MeV/nuc and ULEIS ~0.2--1 MeV/nuc) we have investigated heavy element abundances over a broad energy range in this special set of events. We report the average abundance ratios and the correlations between different ratios in the two energy intervals. Furthermore we compare the results from the two different energy ranges, both statistically and on an event-by-event basis. In addition, we compare the statistical properties observed in the SIS and ULEIS data sets with previously-published results obtained at intermediate energies (~1--3 MeV/nuc) from instruments on ISEE-3 during the maximum of solar cycle 21 (Mason et al. 1986, Reames et al. 1994).

  14. Localization of Passive RFID Tags by Using Broad-Type Multi-Sensing-Range (B-MSR) Method

    NASA Astrophysics Data System (ADS)

    Fujimoto, Manato; Wada, Tomotaka; Inada, Atsuki; Nakamori, Emi; Oda, Yuki; Mutsuura, Kouichi; Okada, Hiromi

    The radio frequency identification (RFID) system has attracting attention as a new identification source that achieves a ubiquitous environment. Each RFID tag has a unique ID code, and is attached on an object whose information it contains. A user reads the unique ID code using RFID readers and obtains information about the object. One of the important applications of RFID technology is the indoor position estimation of RFID tags. It can be applied to navigation systems for people in complex buildings. In this paper, we propose an effective position estimation method named Broad-type Multi-Sensing-Range (B-MSR) method to improve the estimation error of the conventional methods using sensor model. A new reader antenna with two flexible antenna elements is introduced into B-MSR. The distance between two flexible antenna elements can be adjusted. Thus, two kinds of system parameters can be controlled, the distance between two antenna elements and the transmission power of the RFID reader. In this paper, four sensing ranges are settled by controlling the values of two parameters. The performance evaluation shows four characteristics of B-MSR. Firstly, it reduces the initial estimation error. Secondly, it reduces the moving distance. Thirdly, it reduces the number of different sensing points. Fourthly, it shortens the required estimation time.

  15. Experimental study of attenuation and dispersion over a broad frequency range: 2. The universal scaling of polycrystalline materials

    NASA Astrophysics Data System (ADS)

    McCarthy, Christine; Takei, Yasuko; Hiraga, Takehiko

    2011-09-01

    In order to extend the range of conditions that can be obtained in experiments, we have measured the viscoelastic properties of polycrystalline organic borneol, as an analogue to mantle rock. Using a custom fabricated apparatus, the Young's modulus E and attenuation QE-1 were measured accurately over a broad frequency range (10-4 ≤ f (Hz) ≤ 2.15) and at low strain amplitude (10-5-10-6). Creep experiments were performed with the same apparatus to measure the steady state viscosity. Anelasticity and viscosity were measured at high homologous temperatures (T = 22-48°C; T/Tm = 0.61-0.67) and various grain sizes (3-22 μm), the growth of which was controlled by annealing. Using the measured viscosities η and the unrelaxed modulus EU determined from ultrasonic experiments, the frequency of the entire data set was normalized by the Maxwell frequency fM = EU/η, resulting in E and Q-1 master curves. The Q-1 data from previous studies on olivine-dominated samples also collapse onto the same curve when scaled by fM,, demonstrating the universality of anelasticity for polycrystalline materials. The similitude by the Maxwell frequency scaling indicates that the dominant mechanism for the anelasticity observed in this study and in previous studies is diffusionally accommodated grain boundary sliding. A generalized formulation for this similitude is provided to extrapolate the experimental data to velocity and attenuation of seismic shear waves.

  16. Knowing the dense plasma focus - The coming of age (of the PF) with broad-ranging scaling laws

    NASA Astrophysics Data System (ADS)

    Saw, S. H.; Lee, S.

    2017-03-01

    The dense plasma focus is blessed not only with copious multi-radiations ranging from electron and ion beams, x-rays both soft and hard, fusion neutrons D-D and D-T but also with the property of enhanced compression from radiative collapse leading to HED (high energy density) states. The Lee code has been used in extensive systematic numerical experiments tied to reality through fitting with measured current waveforms and verified through comparison of measured and computed yields and measurements of multi-radiation. The studies have led to establishment of scaling laws with respect to storage energy, discharge current and pinch currents for fusion neutrons, characteristic soft x-rays, all-line radiation and ion beams. These are summarized here together with a first-time presentation of a scaling law of radiatively enhanced compression as a function of atomic number of operational gas. This paper emphasizes that such a broad range of scaling laws signals the coming of age of the DPF and presents a reference platform for planning the many potential applications such as in advanced SXR lithography, materials synthesizing and testing, medical isotopes, imaging and energy and high energy density (HED).

  17. Division of labor by dual feedback regulators controls JAK2/STAT5 signaling over broad ligand range

    PubMed Central

    Bachmann, Julie; Raue, Andreas; Schilling, Marcel; Böhm, Martin E; Kreutz, Clemens; Kaschek, Daniel; Busch, Hauke; Gretz, Norbert; Lehmann, Wolf D; Timmer, Jens; Klingmüller, Ursula

    2011-01-01

    Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. The specific contributions of individual feedback regulators, however, remain unclear. Based on extensive time-resolved data sets in primary erythroid progenitor cells, we established a dynamic pathway model to dissect the roles of the two transcriptional negative feedback regulators of the suppressor of cytokine signaling (SOCS) family, CIS and SOCS3, in JAK2/STAT5 signaling. Facilitated by the model, we calculated the STAT5 response for experimentally unobservable Epo concentrations and provide a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. Model predictions show that the two feedbacks CIS and SOCS3 are most effective at different ligand concentration ranges due to their distinct inhibitory mechanisms. This divided function of dual feedback regulation enables control of STAT5 responses for Epo concentrations that can vary 1000-fold in vivo. Our modeling approach reveals dose-dependent feedback control as key property to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations. PMID:21772264

  18. Ultra-broad range organic solid-state laser from a dye-doped holographic grating quasi-waveguide configuration

    NASA Astrophysics Data System (ADS)

    Liu, Minghuan; Liu, Yonggang; Peng, Zenghui; Mu, Quanquan; Cao, Zhaoliang; Lu, Xinghai; Ma, Ji; Xuan, Li

    2017-08-01

    This paper reports the ultra-broad 149.1 nm lasing emission from 573.2 to 722.3 nm using a simple [4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran] (DCM)-doped holographic polymer-dispersed liquid crystal (HPDLC) grating quasi-waveguide configuration by varying the grating period. The lasing emission beams show s-polarization property. The quasi-waveguide structure, which contained the cover glass, the DCM-doped HPDLC grating, the semiconducting polymer film poly[-methoxy-5-(2‧-ethyl-hexyloxy)-1,4-phenylene-vinylene] (MEH-PPV), and the substrate were confirmed to decrease lasing threshold and broaden lasing wavelength. The operational lifetime of the device is 240 000 pulses, which corresponds to an overall laser duration of more than 6 h at a repetition rate of 10 Hz. In addition, the dual-wavelength lasing range from the 8th and 9th order is over 40 nm. The electrical tunability of the dual-wavelength lasing emission is over 1 nm. The experimental results facilitated the decreased lasing threshold and broadened lasing wavelength range of organic solid-state lasers.

  19. Optically stabilized Erbium fiber frequency comb with hybrid mode-locking and a broad tunable range of repetition rate.

    PubMed

    Yang, Honglei; Wu, Xuejian; Zhang, Hongyuan; Zhao, Shijie; Yang, Lijun; Wei, Haoyun; Li, Yan

    2016-12-01

    We present an optically stabilized Erbium fiber frequency comb with a broad repetition rate tuning range based on a hybrid mode-locked oscillator. We lock two comb modes to narrow-linewidth reference lasers in turn to investigate the best performance of control loops. The control bandwidth of fast and slow piezoelectric transducers reaches 70 kHz, while that of pump current modulation with phase-lead compensation is extended to 32 kHz, exceeding laser intrinsic response. Eventually, simultaneous lock of both loops is realized to totally phase-stabilize the comb, which will facilitate precision dual-comb spectroscopy, laser ranging, and timing distribution. In addition, a 1.8-MHz span of the repetition rate is achieved by an automatic optical delay line that is helpful in manufacturing a secondary comb with a similar repetition rate. The oscillator is housed in a homemade temperature-controlled box with an accuracy of ±0.02  K, which not only keeps high signal-to-noise ratio of the beat notes with reference lasers, but also guarantees self-starting at the same mode-locking every time.

  20. High energy storage density over a broad temperature range in sodium bismuth titanate-based lead-free ceramics.

    PubMed

    Yang, Haibo; Yan, Fei; Lin, Ying; Wang, Tong; Wang, Fen

    2017-08-18

    A series of (1-x)Bi0.48La0.02Na0.48Li0.02Ti0.98Zr0.02O3-xNa0.73Bi0.09NbO3 ((1-x)LLBNTZ-xNBN) (x = 0-0.14) ceramics were designed and fabricated using the conventional solid-state sintering method. The phase structure, microstructure, dielectric, ferroelectric and energy storage properties of the ceramics were systematically investigated. The results indicate that the addition of Na0.73Bi0.09NbO3 (NBN) could decrease the remnant polarization (P r ) and improve the temperature stability of dielectric constant obviously. The working temperature range satisfying TCC 150 °C ≤±15% of this work spans over 400 °C with the compositions of x ≥ 0.06. The maximum energy storage density can be obtained for the sample with x = 0.10 at room temperature, with an energy storage density of 2.04 J/cm(3) at 178 kV/cm. In addition, the (1-x)LLBNTZ-xNBN ceramics exhibit excellent energy storage properties over a wide temperature range from room temperature to 90 °C. The values of energy storage density and energy storage efficiency is 0.91 J/cm(3) and 79.51%, respectively, for the 0.90LLBNTZ-0.10NBN ceramic at the condition of 100 kV/cm and 90 °C. It can be concluded that the (1-x)LLBNTZ-xNBN ceramics are promising lead-free candidate materials for energy storage devices over a broad temperature range.

  1. Broad-Range Electrically Tunable Plasmonic Resonances of a Multilayer Coaxial Nanohole Array with an Electroactive Polymer Wrapper.

    PubMed

    Zhou, Ziwei; Yu, Ye; Sun, Ningwei; Möhwald, Helmuth; Gu, Panpan; Wang, Liyan; Zhang, Wei; König, Tobias A F; Fery, Andreas; Zhang, Gang

    2017-10-11

    Plasmonic assemblies featuring high sensitivity that can be readily shifted by external fields are the key for sensitive and versatile sensing devices. In this paper, a novel fast-responsive plasmonic nanocomposite composed of a multilayer nanohole array and a responsive electrochromic polymer is proposed with the plasmonic mode appearance vigorously cycled upon orthogonal electrical stimuli. In this nanocomposite, the coaxially stacked plasmonic nanohole arrays can induce multiple intense Fano resonances, which result from the crosstalk between a broad surface plasmon resonance (SPR) and the designed discrete transmission peaks with ultrahigh sensitivity; the polymer wrapper could provide the sensitive nanohole array with real-time-varied surroundings of refractive indices upon electrical stimuli. Therefore, a pronounced pure electroplasmonic shift up to 72 nm is obtained, which is the largest pure electrotuning SPR range to our knowledge. The stacked nanohole arrays here are also directly used as a working electrode, and they ensure sufficient contact between the working electrode (plasmonic structure) and the electroactive polymer, thus providing considerably improved response speed (within 1 s) for real-time sensing and switching.

  2. Broad-range PCR coupled with mass-spectrometry for the detection of Mycobacterium tuberculosis drug resistance.

    PubMed

    Florea, Dragoş; Oţelea, Dan; Olaru, Ioana D; Hristea, Adriana

    2016-03-01

    The need to limit the spread of drug-resistant Mycobacterium tuberculosis requires rapid detection of resistant strains. The present study aimed to evaluate a commercial assay using broad-range PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) for the rapid detection of isoniazid (INH) and rifampin (RIF) resistance in M. tuberculosis strains isolated from Romanian patients with pulmonary tuberculosis. PCR/ESI-MS was used to detect genotypic resistance to RIF and INH in a panel of 63 M. tuberculosis isolates phenotypically characterized using the absolute concentration method on Löwenstein-Jensen medium. Thirty-eight (60%) strains were susceptible to both drugs, 22 (35%) were RIF and INH resistant, one was INH mono-resistant and two were RIF mono-resistant. The sensitivity for INH and RIF resistance mutations detection were 100% and 92% respectively, with a specificity of more than 95% for each drug. PCR/ESI-MS is a good method for the detection of RIF and INH resistance and might represent an alternative to other rapid diagnostic tests for the detection of genetic markers of resistance in M. tuberculosis isolates.

  3. Tongue Sole CD209: A Pattern-Recognition Receptor that Binds a Broad Range of Microbes and Promotes Phagocytosis.

    PubMed

    Jiang, Shuai; Sun, Li

    2017-09-04

    CD209 is an immune receptor that plays an important role in the initiation of innate immunity and activation of adaptive immunity in mammals. However, much less is known about the immunological function of CD209 in lower vertebrates. In the present study, we examined the immune effect of a CD209 homologue (CsCD209) from the teleost fish tongue sole Cynoglossus semilaevis. CsCD209 possesses a lectin domain that shares high levels of similarity with the lectin domains of human and mouse CD209. CsCD209 expression was most abundant in kidney and blood and was significantly upregulated during bacterial infection. CsCD209 exhibited a subcellular localization mainly on the cell surface of myelomonocytes. Recombinant CsCD209 displayed apparent binding capacities to a broad range of bacteria and fungi, and significantly promoted the phagocytosis of the bound bacteria by C. semilaevis leukocytes. Collectively, the results indicate that teleost CD209 serves as a pattern recognition receptor that exerts an influence on the phagocytosis process during pathogen infections.

  4. Nanohole-based SPR Instruments with Improved Spectral Resolution Quantify a Broad Range of Antibody-Ligand Binding Kinetics

    PubMed Central

    Im, Hyungsoon; Sutherland, Jamie N.; Maynard, Jennifer A.; Oh, Sang-Hyun

    2012-01-01

    We demonstrate an affordable low-noise SPR instrument based on extraordinary optical transmission (EOT) in metallic nanohole arrays and quantify a broad range of antibody-ligand binding kinetics with equilibrium dissociation constants ranging from 200 pM to 40 nM. This nanohole-based SPR instrument is straightforward to construct, align, and operate, since it is built around a standard microscope and a portable fiber-optic spectrometer. The measured refractive index resolution of this platform is 3.1 × 10−6 without on-chip cooling, which is among the lowest reported for SPR sensors based on EOT. This is accomplished via rapid full-spectrum acquisition in 10 milliseconds followed by frame averaging of the EOT spectra, which is made possible by the production of template-stripped gold nanohole arrays with homogeneous optical properties over centimeter-sized areas. Sequential SPR measurements are performed using a 12-channel microfluidic flow cell after optimizing surface modification protocols and antibody injection conditions to minimize mass-transport artifacts. The immobilization of a model ligand, the protective antigen of anthrax on the gold surface, is monitored in real-time with a signal-to-noise ratio of ~860. Subsequently, real-time binding kinetic curves were measured quantitatively between the antigen and a panel of small, 25 kDa single-chain antibodies at concentrations down to 1 nM. These results indicate that nanohole-based SPR instruments have potential for quantitative antibody screening and as a general-purpose platform for integrating SPR sensors with other bioanalytical tools. PMID:22235895

  5. Complete Genome Sequence of the Broad-Host-Range Vibriophage KVP40: Comparative Genomics of a T4-Related Bacteriophage

    PubMed Central

    Miller, Eric S.; Heidelberg, John F.; Eisen, Jonathan A.; Nelson, William C.; Durkin, A. Scott; Ciecko, Ann; Feldblyum, Tamara V.; White, Owen; Paulsen, Ian T.; Nierman, William C.; Lee, Jong; Szczypinski, Bridget; Fraser, Claire M.

    2003-01-01

    The complete genome sequence of the T4-like, broad-host-range vibriophage KVP40 has been determined. The genome sequence is 244,835 bp, with an overall G+C content of 42.6%. It encodes 386 putative protein-encoding open reading frames (CDSs), 30 tRNAs, 33 T4-like late promoters, and 57 potential rho-independent terminators. Overall, 92.1% of the KVP40 genome is coding, with an average CDS size of 587 bp. While 65% of the CDSs were unique to KVP40 and had no known function, the genome sequence and organization show specific regions of extensive conservation with phage T4. At least 99 KVP40 CDSs have homologs in the T4 genome (Blast alignments of 45 to 68% amino acid similarity). The shared CDSs represent 36% of all T4 CDSs but only 26% of those from KVP40. There is extensive representation of the DNA replication, recombination, and repair enzymes as well as the viral capsid and tail structural genes. KVP40 lacks several T4 enzymes involved in host DNA degradation, appears not to synthesize the modified cytosine (hydroxymethyl glucose) present in T-even phages, and lacks group I introns. KVP40 likely utilizes the T4-type sigma-55 late transcription apparatus, but features of early- or middle-mode transcription were not identified. There are 26 CDSs that have no viral homolog, and many did not necessarily originate from Vibrio spp., suggesting an even broader host range for KVP40. From these latter CDSs, an NAD salvage pathway was inferred that appears to be unique among bacteriophages. Features of the KVP40 genome that distinguish it from T4 are presented, as well as those, such as the replication and virion gene clusters, that are substantially conserved. PMID:12923095

  6. Genome sequence and analysis of a broad-host range lytic bacteriophage that infects the Bacillus cereus group

    PubMed Central

    2013-01-01

    Background Comparatively little information is available on members of the Myoviridae infecting low G+C content, Gram-positive host bacteria of the family Firmicutes. While numerous Bacillus phages have been isolated up till now only very few Bacillus cereus phages have been characterized in detail. Results Here we present data on the large, virulent, broad-host-range B. cereus phage vB_BceM_Bc431v3 (Bc431v3). Bc431v3 features a 158,618 bp dsDNA genome, encompassing 239 putative open reading frames (ORFs) and, 20 tRNA genes encoding 17 different amino acids. Since pulsed-field gel electrophoresis indicated that the genome of this phage has a mass of 155-158 kb Bc431v3 DNA appears not to contain long terminal repeats that are found in the genome of Bacillus phage SPO1. Conclusions Bc431v3 displays significant sequence similarity, at the protein level, to B. cereus phage BCP78, Listeria phage A511 and Enterococcus phage ØEF24C and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Based on these data we suggest that Bc431v3 should be included as a member of the Spounavirinae; however, because of all the diverse taxonomical information has been addressed recently, it is difficult to determine the genus. The Bc431v3 phage contains some highly unusual genes such as gp143 encoding putative tRNAHis guanylyltransferase. In addition, it carries some genes that appear to be related to the host sporulation regulators. These are: gp098, which encodes a putative segregation protein related to FstK/SpoIIIE DNA transporters; gp105, a putative segregation protein; gp108, RNA polymerase sigma factor F/B; and, gp109 encoding RNA polymerase sigma factor G. PMID:23388049

  7. Broad-Range Detection of Microorganisms Directly from Bronchoalveolar Lavage Specimens by PCR/Electrospray Ionization-Mass Spectrometry

    PubMed Central

    Ullberg, Måns; Lüthje, Petra; Mölling, Paula; Strålin, Kristoffer

    2017-01-01

    The clinical demand on rapid microbiological diagnostic is constantly increasing. PCR coupled to electrospray ionization-mass spectrometry, PCR/ESI-MS, offers detection and identification of over 750 bacteria and Candida species directly from clinical specimens within 6 hours. In this study, we investigated the clinical performance of the IRIDICA BAC LRT Assay for detection of bacterial pathogens in 121 bronchoalveolar lavage (BAL) samples that were received consecutively at our bacterial laboratory for BAL culture. Commensal or pathogenic microorganisms were detected in 118/121 (98%) BAL samples by PCR/ESI-MS, while in 104/121 (86%) samples by routine culture (P<0.01). Detection of potentially pathogenic microorganisms by PCR/ESI-MS was evaluated in comparison with conventional culture-based or molecular methods. The agreement between positive findings was overall good. Most Staphylococcus aureus-positive PCR/ESI-MS results were confirmed by culture or species-specific PCR (27/33, 82%). The identity of Streptococcus pneumoniae could however be confirmed for only 6/17 (35%) PCR/ESI-MS-positive samples. Non-cultivable and fastidious pathogens, which were not covered by standard culture procedures were readily detected by PCR/ESI-MS, including Legionella pneumophila, Bordetella pertussis, Norcadia species and Mycoplasma pneumoniae. In conclusion, PCR/ESI-MS detected a broad range of potential pathogens with equal or superior sensitivity compared to conventional methods within few hours directly from BAL samples. This novel method might thus provide a relevant tool for diagnostics in critically ill patients. PMID:28085931

  8. Why to measure a broad range of city sizes? Analysis of globally pooled data of urban GHG measurements for sustainability

    NASA Astrophysics Data System (ADS)

    Rybski, Diego; Sterzel, Till; Reusser, Dominik E.; Fichter, Christina; Kropp, Jürgen P.

    2013-04-01

    We have assembled a database of urban GHG emissions from various published sources, including about 200 cities globally. Analyzing this CO2 emission inventory from multiple countries we find power-law relations between the emissions and city size, measured in population. The results suggest that in developing countries large cities emit more CO2 per capita compared to small cities, i.e. they tend to comprise super-linear correlations. For developed countries the results suggest the opposite, i.e. linear or sub-linear correlations, implying better efficiency of large cities. We derive how the total emissions of an entire country relate with the power-law correlations and find that the size of the most populated city is dominating in the case of linear and super-linear correlations, while a transition occurs to sub-linear correlations, where the size of the largest city has no influence. It is important to further substantiate an overview of city emission inventories across a broad range of city sizes and types to further clarify the complex relationships between cities and GHG emissions. On the one hand, we propose a minimum set of meta-information to be reported together with the emission inventories, e.g. for determining comparability among inventories. On the other hand, we propose to fill evident gaps with respect to regions (e.g. sub-Saharan African and South American cities) and types of cities (e.g. small medium and low-income country cities) to allow for a better global overview of city sizes, income, and emissions. We conclude that from the climate change mitigation point of view, urbanization is desirable in developed countries and should be avoided in developing countries, if effinciency increasing mechanisms can not be established. More data acquisition is needed to support our empirical findings.

  9. Forest response to elevated CO2 is conserved across a broad range of productivity

    SciTech Connect

    Norby, Richard J; DeLucia, E. H.; Gielen, Birgit; Califapietra, Carlo; Giardina, Christian P; King, John S.; Childs, Joanne; McCarthy, Heather R; Moore, D J; Ceulemans, Reinhart; DeAngelis, Paolo; Finzi, Adrien C; Karnosky, David; Kubiske, Mark E; Lukac, Martin; Pregitzer, Kurt; Scarascia-Mugnozza, Giuseppe E; Schlesinger, William H; Oren, Ram

    2005-11-01

    Climate change predictions derived from coupled carbon-climate models are highly dependent on assumptions about feedbacks between the biosphere and atmosphere. One critical feedback occurs if C uptake by the biosphere increases in response to the fossil-fuel driven increase in atmospheric [CO{sub 2}] ('CO{sub 2} fertilization'), thereby slowing the rate of increase in atmospheric [CO{sub 2}]. Carbon exchanges between the terrestrial biosphere and atmosphere are often first represented in models as net primary productivity (NPP). However, the contribution of CO{sub 2} fertilization to the future global C cycle has been uncertain, especially in forest ecosystems that dominate global NPP, and models that include a feedback between terrestrial biosphere metabolism and atmospheric [CO{sub 2}] are poorly constrained by experimental evidence. We analyzed the response of NPP to elevated CO{sub 2} ({approx}550 ppm) in four free-air CO{sub 2} enrichment experiments in forest stands. We show that the response of forest NPP to elevated [CO{sub 2}] is highly conserved across a broad range of productivity, with a stimulation at the median of 23 {+-} 2%. At low leaf area indices, a large portion of the response was attributable to increased light absorption, but as leaf area indices increased, the response to elevated [CO{sub 2}] was wholly caused by increased light-use efficiency. The surprising consistency of response across diverse sites provides a benchmark to evaluate predictions of ecosystem and global models and allows us now to focus on unresolved questions about carbon partitioning and retention, and spatial variation in NPP response caused by availability of other growth limiting resources.

  10. Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses.

    PubMed Central

    Pruss, G; Ge, X; Shi, X M; Carrington, J C; Bowman Vance, V

    1997-01-01

    Synergistic viral diseases of higher plants are caused by the interaction of two independent viruses in the same host and are characterized by dramatic increases in symptoms and in accumulation of one of the coinfecting viruses. In potato virus X (PVX)/potyviral synergism, increased pathogenicity and accumulation of PVX are mediated by the expression of potyviral 5' proximal sequences encoding P1, the helper component proteinase (HC-Pro), and a fraction of P3. Here, we report that the same potyviral sequence (termed P1/HC-Pro) enhances the pathogenicity and accumulation of two other heterologous viruses: cucumber mosaic virus and tobacco mosaic virus. In the case of PVX-potyviral synergism, we show that the expression of the HC-Pro gene product, but not the RNA sequence itself, is sufficient to induce the increase in PVX pathogenicity and that both P1 and P3 coding sequences are dispensable for this aspect of the synergistic interaction. In protoplasts, expression of the potyviral P1/HC-Pro region prolongs the accumulation of PVX (-) strand RNA and transactivates expression of a reporter gene from a PVX subgenomic promoter. Unlike the synergistic enhancement of PVX pathogenicity, which requires only expression of HC-Pro, the enhancement of PVX (-) strand RNA accumulation in protoplasts is significantly greater when the entire P1/HC-Pro sequence is expressed. These results indicate that the potyviral P1/HC-Pro region affects a step in disease development that is common to a broad range of virus infections and suggest a mechanism involving transactivation of viral replication. PMID:9212462

  11. Silver-Russell patients showing a broad range of ICR1 and ICR2 hypomethylation in different tissues.

    PubMed

    Begemann, M; Spengler, S; Kanber, D; Haake, A; Baudis, M; Leisten, I; Binder, G; Markus, S; Rupprecht, T; Segerer, H; Fricke-Otto, S; Mühlenberg, R; Siebert, R; Buiting, K; Eggermann, T

    2011-07-01

    In all known congenital imprinting disorders an association with aberrant methylation or mutations at specific loci was well established. However, several patients with transient neonatal diabetes mellitus (TNDM), Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS) exhibiting multilocus hypomethylation (MLH) have meanwhile been described. Whereas TNDM patients with MLH show clinical symptoms different from carriers with isolated 6q24 aberrations, MLH carriers diagnosed as BWS or SRS present only the syndrome-specific features. Interestingly, SRS and BWS patients with nearly identical MLH patterns in leukocytes have been identified. We now report on the molecular findings in DNA in three SRS patients with hypomethylation of both 11p15 imprinted control regions (ICRs) in leukocytes. One patient was a monozygotic (MZ) twin, another was a triplet. While the hypomethylation affected both oppositely imprinted 11p15 ICRs in leukocytes, in buccal swab DNA only the ICR1 hypomethylation was visible in two of our patients. In the non-affected MZ twin of one of these patients, aberrant methylation was also present in leukocytes but neither in buccal swab DNA nor in skin fibroblasts. Despite mutation screening of several factors involved in establishment and maintenance of methylation marks including ZFP57, MBD3, DNMT1 and DNMT3L the molecular clue for the ICR1/ICR2 hypomethylation in our patients remained unclear. Furthermore, the reason for the development of the specific SRS phenotype is not obvious. In conclusion, our data reflect the broad range of epimutations in SRS and illustrate that an extensive molecular and clinical characterization of patients is necessary.

  12. Cyclic loading experiments to measure material response over a broad frequency range: from tickling of rocks to squeezing of moons

    NASA Astrophysics Data System (ADS)

    McCarthy, C.; Takei, Y.; Cooper, R. F.; Savage, H. M.

    2014-12-01

    Seismology provides powerful methods for imaging the interior of the Earth, not only through differences in seismic velocities, but also through attenuation contrasts. As seismic waves travel through the Earth they are attenuated in accordance with the viscoelastic properties of the material through which they pass. With proper constraints, we will someday be able to use seismic attenuation data as a prospecting tool to determine the grain size, temperature, pressure, melt content, and water content of the material along the ray path. Furthermore, it should be possible to determine active deformation structure, such as crystallographic preferred orientations that form in response to far-field natural tectonic loading. Laboratory studies are striving to provide these needed constraints. Using analogues to mantle rock, we isolate and scrutinize the physics of how microstructural elements affect macroscopic properties of attenuation and steady-state viscosity. An organic analogue, borneol, was used to measure the effects of grain size, temperature, and melt content over a broad frequency range. In these experiments, grain boundary processes were found to play a major role. Polycrystalline ice, which can be considered a rock analogue, has been used to explore the effect of accumulated strain on attenuation, particularly in material that is actively deforming via dislocation creep. Here, defect concentration and substructure are important. I will discuss the use of cyclic loading experiments on borneol and on polycrystalline ice to probe material response from seismic to tidal frequencies, from 10 Hz to 10-4 Hz respectively. These experiments, then, inform our knowledge of viscoelastic behavior of geologic materials at not only seismic frequencies, but also the tidal forcing frequencies experienced by tidewater glaciers and icy satellites.

  13. [Isolation and characterization of petroleum catabolic broad-host-range plasmids from Shen-Fu wastewater irrigation zone].

    PubMed

    Wang, Ya-Fei; Wang, Ya-Fei; Li, Hui; Li, Xiao-Bin

    2013-11-01

    Based on triparental mating, we isolated a total of eight broad host range (BHR) petroleum hydrocarbon catabolic plasmids from the soils, sediments, and wastewater samples in the Shen-Fu irrigation zone. The antibiotic resistance of the plasmids was tested, and then, the plasmids were transferred to Escherichia coli EC100. The plasmids carrying no antibiotic resistance were tagged by miniTn5 transposon consisting of antibiotic resistant genes. The PCR-based incompatibility test revealed that the pS3-2C and pS4-6G belonged to Inc P group, the pS3-2G, pW22-3G, and pA15-7G belonged to Inc N group, the pS7-2G was identified as Inc W plasmid, and the pA23-1G and pA10-1C were placed into Inc Q group. By adopting the reported PCR amplification methods of petroleum hydrocarbon-degrading catabolic genes, the petroleum-degrading capability of these BHR plasmids were preliminarily analyzed. The plasmids pS3-2G, pS7-2G, pA23-1G, pW22-3G, and pA10-1C carried aromatic ring- hydroxylating dioxygenase gene phdA and toluene monooxygenase gene touA; the plasmid pA15-7G carried touA and toluene dioxygenase gene tod; the plasmid pS3-2C carried ben, phdA, and tod; whereas the pS4-6G only carried ben. The host range test showed that all the isolated plasmids except pS3-2C could be transferred and maintained stably in the representative strains Agrobacterium tumefaciens C58, Cupriavidus necator JMP228, and E. coli EC100 of the alpha-, beta-, and gamma-Proteobacteria, respectively.

  14. Broad-range PCR in the identification of bacterial and fungal pathogens from positive blood culture bottles: a sequencing approach.

    PubMed

    Morinaga, Yoshitomo; Yanagihara, Katsunori

    2015-01-01

    Rapid identification of causative bacteria in patients with sepsis can contribute to appropriate selection of antibiotics and improvement of patients' prognosis. Genotypic identification is an emerging technology that may provide an alternative method to, or complement, established phenotypic identification procedures.Sequence analysis of the 16S rRNA gene is a widely accepted tool for molecular identification of bacteria. Pyrosequencing is a DNA sequencing technique that is based on the detection of pyrophosphate that is released during DNA synthesis. Pyrosequencing can provide sequence information rapidly by reading short sequences; therefore, it may contribute to a rapid identification and lead to a great help in improving the outcome of sepsis. The DNA pyrosequencing-based identification from positive blood culture samples basically consisted of the following four steps: (1) DNA extraction, (2) amplification of target genes, (3) DNA pyrosequencing, and (4) homology searching.

  15. Diversity of thermophiles in a Malaysian hot spring determined using 16S rRNA and shotgun metagenome sequencing.

    PubMed

    Chan, Chia Sing; Chan, Kok-Gan; Tay, Yea-Ling; Chua, Yi-Heng; Goh, Kian Mau

    2015-01-01

    The Sungai Klah (SK) hot spring is the second hottest geothermal spring in Malaysia. This hot spring is a shallow, 150-m-long, fast-flowing stream, with temperatures varying from 50 to 110°C and a pH range of 7.0-9.0. Hidden within a wooded area, the SK hot spring is continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC). In this study, a sample taken from the middle of the stream was analyzed at the 16S rRNA V3-V4 region by amplicon metagenome sequencing. Over 35 phyla were detected by analyzing the 16S rRNA data. Firmicutes and Proteobacteria represented approximately 57% of the microbiome. Approximately 70% of the detected thermophiles were strict anaerobes; however, Hydrogenobacter spp., obligate chemolithotrophic thermophiles, represented one of the major taxa. Several thermophilic photosynthetic microorganisms and acidothermophiles were also detected. Most of the phyla identified by 16S rRNA were also found using the shotgun metagenome approaches. The carbon, sulfur, and nitrogen metabolism within the SK hot spring community were evaluated by shotgun metagenome sequencing, and the data revealed diversity in terms of metabolic activity and dynamics. This hot spring has a rich diversified phylogenetic community partly due to its natural environment (plant litter, high TOC, and a shallow stream) and geochemical parameters (broad temperature and pH range). It is speculated that symbiotic relationships occur between the members of the community.

  16. FTO gene associated fatness in relation to body fat distribution and metabolic traits throughout a broad range of fatness.

    PubMed

    Kring, Sofia I I; Holst, Claus; Zimmermann, Esther; Jess, Tine; Berentzen, Tina; Toubro, Søren; Hansen, Torben; Astrup, Arne; Pedersen, Oluf; Sørensen, Thorkild I A

    2008-08-13

    A common single nucleotide polymorphism (SNP) of FTO (rs9939609, T/A) is associated with total body fatness. We investigated the association of this SNP with abdominal and peripheral fatness and obesity-related metabolic traits in middle-aged men through a broad range of fatness present already in adolescence. Obese young Danish men (n = 753, BMI > or = 31.0 kg/m(2)) and a randomly selected group (n = 879) from the same population were examined in three surveys (mean age 35, 46 and 49 years, respectively). The traits included anthropometrics, body composition, oral glucose tolerance test, blood lipids, blood pressure, fibrinogen and aspartate aminotransferase. Logistic regression analysis was used to assess the age-adjusted association between the phenotypes and the odds ratios for the FTO rs9939609 (TT and TA genotype versus the AA genotype), for anthropometrics and body composition estimated per unit z-score. BMI was strongly associated with the AA genotype in all three surveys: OR = 1.17, p = 1.1*10(-6), OR = 1.20, p = 1.7*10(-7), OR = 1.17, p = 3.4*10(-3), respectively. Fat body mass index was also associated with the AA genotype (OR = 1.21, p = 4.6*10(-7) and OR = 1.21, p = 1.0*10(-3)). Increased abdominal fatness was associated with the AA genotype when measured as waist circumference (OR = 1.21, p = 2.2*10(-6) and OR = 1.19, p = 5.9*10(-3)), sagittal abdominal diameter (OR = 1.17, p = 1.3*10(-4) and OR = 1.18, p = 0.011) and intra-abdominal adipose tissue (OR = 1.21, p = 0.005). Increased peripheral fatness measured as hip circumference (OR = 1.19, p = 1.3*10(-5) and OR = 1.18, p = 0.004) and lower body fat mass (OR = 1.26, p = 0.002) was associated with the AA genotype. The AA genotype was significantly associated with decreased Stumvoll insulin sensitivity index (OR = 0.93, p = 0.02) and with decreased non-fasting plasma HDL-cholesterol (OR = 0.57, p = 0.037), but not with any other of the metabolic traits. However, all significant results for both body

  17. Aminoglycoside antibiotics: A-site specific binding to 16S

    NASA Astrophysics Data System (ADS)

    Baker, Erin Shammel; Dupuis, Nicholas F.; Bowers, Michael T.

    2009-06-01

    The A-site of 16S rRNA, which is a part of the 30S ribosomal subunit involved in prokaryotic translation, is a well known aminoglycoside binding site. Full characterization of the conformational changes undergone at the A-site upon aminoglycoside binding is essential for development of future RNA/drug complexes; however, the massiveness of 16S makes this very difficult. Recently, studies have found that a 27 base RNA construct (16S27) that comprises the A-site subdomain of 16S behaves similarly to the whole A-site domain. ESI-MS, ion mobility and molecular dynamics methods were utilized in this study to analyze the A-site of 16S27 before and after the addition of ribostamycin (R), paromomycin (P) and lividomycin (L). The ESI mass spectrum for 16S27 alone illustrated both single-stranded 16S27 and double-stranded (16S27)2 complexes. Upon aminoglycoside addition, the mass spectra showed that only one aminoglycoside binds to 16S27, while either one or two bind to (16S27)2. Ion mobility measurements and molecular dynamics calculations were utilized in determining the solvent-free structures of the 16S27 and (16S27)2 complexes. These studies found 16S27 in a hairpin conformation while (16S27)2 existed as a cruciform. Only one aminoglycoside binds to the single A-site of the 16S27 hairpin and this attachment compresses the hairpin. Since two A-sites exist for the (16S27)2 cruciform, either one or two aminoglycosides may bind. The aminoglycosides compress the A-sites causing the cruciform with just one aminoglycoside bound to be larger than the cruciform with two bound. Non-specific binding was not observed in any of the aminoglycoside/16S27 complexes.

  18. Role of the parCBA Operon of the Broad-Host-Range Plasmid RK2 in Stable Plasmid Maintenance

    PubMed Central

    Easter, Carla L.; Schwab, Helmut; Helinski, Donald R.

    1998-01-01

    The par region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons, parCBA and parDE, and a cis-acting site. parDE encodes a postsegregational killing system, and parCBA encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the parCBA region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of Escherichia coli and two strains of Pseudomonas aeruginosa. The intact parCBA operon was effective in stabilizing a conjugation-defective RK2 derivative in E. coli MC1061K and RR1 but was relatively ineffective in E. coli MV10Δlac. In the two strains in which the parCBA operon was effective, deletions in parB, parC, or both parB and parC caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the parA gene resulted in a much greater loss of parCBA activity. For P. aeruginosa PAO1161Rifr, the parCBA operon provided little if any plasmid stability, but for P. aeruginosa PAC452Rifr, the RK2 plasmid was stabilized to a substantial extent by parCBA. With this latter strain, parA and res alone were sufficient for stabilization. The cer resolvase system of plasmid ColE1 and the loxP/Cre system of plasmid P1 were tested in comparison with the parCBA operon. We found that, not unlike what was previously observed with MC1061K, cer failed to stabilize the RK2 plasmid with par deletions in strain MV10Δlac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The loxP/Cre system, on the other hand, was very effective in stabilizing the plasmid in all three E. coli strains. These observations indicate that the parA gene, along with its res site, exhibits a significant level of plasmid stabilization in the absence of the parC and

  19. Disentangling climatic versus biotic drivers of tree range constraints: Broad scale tradeoffs between climate and competion rarely explain local range boundaries

    NASA Astrophysics Data System (ADS)

    Anderegg, L. D. L.; Hillerislambers, J.

    2016-12-01

    Accurate prediction of climatically-driven range shifts requires knowledge of the dominant forces constraining species ranges, because climatically controlled range boundaries will likely behave differently from biotically controlled range boundaries in a changing climate. Yet the roles of climatic constraints (due to species physiological tolerance) versus biotic constraints (caused by species interactions) on geographic ranges are largely unknown, infusing large uncertainty into projections of future range shifts. Plant species ranges across strong climatic gradients such as elevation gradients are often assumed to represent a tradeoff between climatic constraints on the harsh side of the range and biotic constraints (often competitive constraints) on the climatically benign side. To test this assumption, we collected tree cores from across the elevational range of the three dominant tree species inhabiting each of three climatically disparate mountain slopes and assessed climatic versus competitive constraints on growth at each species' range margins. Across all species and mountains, we found evidence for a tradeoff between climatic and competitve growth constraints. We also found that some individual species did show an apparent trade-off between a climatic constraint at one range margin and a competitive constraint at the other. However, even these simple elevation gradients resulted in complex interactions between temperature, moisture, and competitive constraints such that a climate-competition tradeoff did not explain range constraints for many species. Our results suggest that tree species can be constrained by a simple trade-off between climate and competition, but that the intricacies of real world climate gradients complicate the application of this theory even in apparently harsh environments, such as near high elevation tree line.

  20. Broad-range neutralizing anti-influenza A human monoclonal antibodies: new perspectives in therapy and prophylaxis.

    PubMed

    Clementi, Nicola; Criscuolo, Elena; Castelli, Matteo; Clementi, Massimo

    2012-10-01

    Broadly neutralizing monoclonal antibodies (mAbs) directed against different subtypes of influenza A viruses are novel tools for the potential development of effective anti-influenza prophylactic and therapeutic strategies. In both cases, the main candidates for passive transfer and new vaccine development are represented by protective mAbs directed against influenza hemagglutinin (HA). A large number of mAbs directed against influenza HA has been developed to date. However, even if they can be useful and contribute to develop new vaccinal strategies, only few of them can be a good candidate for human administration. In this review, we will describe the most relevant human mAb directed against influenza HA able to recognize highly divergent influenza isolates and possibly useful for human therapy and prophylaxis.

  1. Bio-inspired anti-oil-fouling chitosan-coated mesh for oil/water separation suitable for broad pH range and hyper-saline environments.

    PubMed

    Zhang, Shiyan; Lu, Fei; Tao, Lei; Liu, Na; Gao, Changrui; Feng, Lin; Wei, Yen

    2013-11-27

    Here, we report a bio-inspired chitosan (CS)-based mesh with high separation efficiency, oil-fouling repellency, and stability in a complex liquid environment. The surface of the CS coating maintains underwater superoleophobicity and low oil adhesion (<1 μN) in pure water and hyper-saline solutions, and it can keep stable special wettability in broad pH range environments after the CS mesh is fully cross-linked with glutaraldehyde and then reduced by sodium borohydride to form a stable carbon-nitrogen single bond. The separation process is solely gravity-driven, and the mesh can separate a range of different oil/water mixtures with >99% separation efficiency in hyper-saline and broad pH range conditions. We envision that such a separation method will be useful in oil spill cleanup and industrial oily wastewater treatment in extreme environments.

  2. Optoinjection for efficient targeted delivery of a broad range of compounds and macromolecules into diverse cell types

    NASA Astrophysics Data System (ADS)

    Clark, Imram; Hanania, Elie G.; Stevens, Janine; Gallina, Marijo; Fieck, Annabeth; Brandes, Rolf; Palsson, Bernhard O.; Koller, Manfred R.

    2006-01-01

    Efficient delivery of compounds and macromolecules into living cells is essential in many fields including basic research, applied drug discovery, and clinical gene therapy. Unfortunately, current delivery methods, such as cationic lipids and electroporation, are limited by the types of macromolecules and cells that can be employed, poor efficiency, and/or cell toxicity. To address these issues, novel methods were developed based on laser-mediated delivery of macromolecules into cells through optoinjection. An automated high-throughput instrument, the laser-enabled analysis and processing (LEAPTM) system, was utilized to elucidate and optimize several parameters that influence optoinjection efficiency and toxicity. Techniques employing direct cell irradiation (i.e., targeted to specific cell coordinates) and grid-based irradiation (i.e., without locating individual cells) were both successfully developed. With both techniques, it was determined that multiple, sequential low radiant exposures produced more favorable results than a single high radiant exposure. Various substances were efficiently optoinjected-including ions, small molecules, dextrans, siRNAs (small interfering RNAs), plasmids, proteins, and semiconductor nanocrystals-into numerous cell types. Notably, cells refractory to traditional delivery methods were efficiently optoinjected with lower toxicity. We establish the broad utility of optoinjection, and furthermore, are the first to demonstrate its implementation in an automated, high-throughput manner.

  3. Derivatives of usnic acid inhibit broad range of influenza viruses and protect mice from lethal influenza infection.

    PubMed

    Shtro, A A; Zarubaev, V V; Luzina, O A; Sokolov, D N; Salakhutdinov, N F

    2015-08-01

    Influenza is a disease of significant morbidity and mortality, the number of anti-influenza drugs is small; many of them stimulate the appearance of resistant strains. In this work, we demonstrate activity of some usnic acid (UA) derivatives against influenza virus in vitro and in vivo. Organic synthesis was used to prepare compounds. Antiviral activity of the compounds in vitro was evaluated by their ability to decrease the virus titer on Madin-Darby Canine Kidney cells. In vivo activity was evaluated by decrease of mortality and index of protection. Compounds were tested against a broad spectrum of influenza virus strains and showed activity against all used strains. One compound, [5] (valine enamine of UA), also significantly reduced lethality of infected animals and does not give rise to the appearance of resistant strains. Additional studies showed that hepatotoxicity of compound [5] is reduced comparatively to UA. Our results suggest that valine enamine of UA could be a potential candidate for the development of a new anti-influenza therapy. © The Author(s) 2016.

  4. Leaf hydraulic vulnerability to drought is linked to site water availability across a broad range of species and climates

    USDA-ARS?s Scientific Manuscript database

    Background and Aims: Vulnerability of the leaf hydraulic pathway to water-stress-induced dysfunction is a key component of drought tolerance in plants and may be important in defining species’ climatic range. However, the generality of the association between leaf hydraulic vulnerability and climate...

  5. LTP in Hippocampal Area CA1 Is Induced by Burst Stimulation over a Broad Frequency Range Centered around Delta

    ERIC Educational Resources Information Center

    Grover, Lawrence M.; Kim, Eunyoung; Cooke, Jennifer D.; Holmes, William R.

    2009-01-01

    Long-term potentiation (LTP) is typically studied using either continuous high-frequency stimulation or theta burst stimulation. Previous studies emphasized the physiological relevance of theta frequency; however, synchronized hippocampal activity occurs over a broader frequency range. We therefore tested burst stimulation at intervals from 100…

  6. LTP in Hippocampal Area CA1 Is Induced by Burst Stimulation over a Broad Frequency Range Centered around Delta

    ERIC Educational Resources Information Center

    Grover, Lawrence M.; Kim, Eunyoung; Cooke, Jennifer D.; Holmes, William R.

    2009-01-01

    Long-term potentiation (LTP) is typically studied using either continuous high-frequency stimulation or theta burst stimulation. Previous studies emphasized the physiological relevance of theta frequency; however, synchronized hippocampal activity occurs over a broader frequency range. We therefore tested burst stimulation at intervals from 100…

  7. C16S - a Hidden Markov Model based algorithm for taxonomic classification of 16S rRNA gene sequences.

    PubMed

    Ghosh, Tarini Shankar; Gajjalla, Purnachander; Mohammed, Monzoorul Haque; Mande, Sharmila S

    2012-04-01

    Recent advances in high throughput sequencing technologies and concurrent refinements in 16S rDNA isolation techniques have facilitated the rapid extraction and sequencing of 16S rDNA content of microbial communities. The taxonomic affiliation of these 16S rDNA fragments is subsequently obtained using either BLAST-based or word frequency based approaches. However, the classification accuracy of such methods is observed to be limited in typical metagenomic scenarios, wherein a majority of organisms are hitherto unknown. In this study, we present a 16S rDNA classification algorithm, called C16S, that uses genus-specific Hidden Markov Models for taxonomic classification of 16S rDNA sequences. Results obtained using C16S have been compared with the widely used RDP classifier. The performance of C16S algorithm was observed to be consistently higher than the RDP classifier. In some scenarios, this increase in accuracy is as high as 34%. A web-server for the C16S algorithm is available at http://metagenomics.atc.tcs.com/C16S/.

  8. Leaf hydraulic vulnerability to drought is linked to site water availability across a broad range of species and climates

    PubMed Central

    Blackman, Chris J.; Gleason, Sean M.; Chang, Yvonne; Cook, Alicia M.; Laws, Claire; Westoby, Mark

    2014-01-01

    Background and Aims Vulnerability of the leaf hydraulic pathway to water-stress-induced dysfunction is a key component of drought tolerance in plants and may be important in defining species' climatic range. However, the generality of the association between leaf hydraulic vulnerability and climate across species and sites remains to be tested. Methods Leaf hydraulic vulnerability to drought (P50leaf, the water potential inducing 50 % loss in hydraulic function) was measured in a diverse group of 92 woody, mostly evergreen angiosperms from sites across a wide range of habitats. These new data together with some previously published were tested against key climate indices related to water availability. Differences in within-site variability in P50leaf between sites were also examined. Key Results Values of hydraulic vulnerability to drought in leaves decreased strongly (i.e. became more negative) with decreasing annual rainfall and increasing aridity across sites. The standard deviation in P50leaf values recorded within each site was positively correlated with increasing aridity. P50leaf was also a good indicator of the climatic envelope across each species' distributional range as well as their dry-end distributional limits within Australia, although this relationship was not consistently detectable within sites. Conclusions The findings indicate that species sorting processes have influenced distributional patterns of P50leaf across the rainfall spectrum, but alternative strategies for dealing with water deficit exist within sites. The strong link to aridity suggests leaf hydraulic vulnerability may influence plant distributions under future climates. PMID:25006181

  9. Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes

    SciTech Connect

    Igarashi, Aki; Yamagata, Kousuke; Sugai, Tomokazu; Takahashi, Yukari; Sugawara, Emiko; Tamura, Akihiro; Yaegashi, Hajime; Yamagishi, Noriko; Takahashi, Tsubasa; Isogai, Masamichi; Takahashi, Hideki; Yoshikawa, Nobuyuki

    2009-04-10

    Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to silence a meristem gene, proliferating cell nuclear antigen and disease resistant N gene in tobacco and RCY1 gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad range of plant species.

  10. Promoters of the Broad Host Range Plasmid Rk2: Analysis of Transcription (Initiation) in Five Species of Gram-Negative Bacteria

    PubMed Central

    Greener, A.; Lehman, S. M.; Helinski, D. R.

    1992-01-01

    A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined. PMID:1732166

  11. Can job redesign interventions influence a broad range of employee outcomes by changing multiple job characteristics? A quasi-experimental study.

    PubMed

    Holman, David; Axtell, Carolyn

    2016-07-01

    Many job redesign interventions are based on a multiple mediator-multiple outcome model in which the job redesign intervention indirectly influences a broad range of employee outcomes by changing multiple job characteristics. As this model remains untested, the aim of this study is to test a multiple mediator-multiple outcome model of job redesign. Multilevel analysis of data from a quasi-experimental job redesign intervention in a call center confirmed the hypothesized model and showed that the job redesign intervention affected a broad range of employee outcomes (i.e., employee well-being, psychological contract fulfillment, and supervisor-rated job performance) through changes in 2 job characteristics (i.e., job control and feedback). The results provide further evidence for the efficacy and mechanisms of job redesign interventions. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

  12. Comparison of PCR-Electrospray Ionization Mass Spectrometry with 16S rRNA PCR and Amplicon Sequencing for Detection of Bacteria in Excised Heart Valves

    PubMed Central

    Peeters, Bart; Herijgers, Paul; Beuselinck, Kurt; Peetermans, Willy E.; Herregods, Marie-Christin

    2016-01-01

    Identification of the causative pathogen of infective endocarditis (IE) is crucial for adequate management and therapy. A broad-range PCR-electrospray ionization mass spectrometry (PCR-ESI-MS) technique was compared with broad-spectrum 16S rRNA PCR and amplicon sequencing (16S rRNA PCR) for the detection of bacterial pathogens in 40 heart valves obtained from 34 definite infective endocarditis patients according to the modified Duke criteria and six nonendocarditis patients. Concordance between the two molecular techniques was 98% for being positive or negative, 97% for concordant identification up to the genus level, and 77% for concordant identification up to the species level. Sensitivity for detecting the causative pathogen (up to the genus level) in excised heart valves was 88% for 16S rRNA PCR and 85% for PCR-ESI-MS; the specificity was 83% for both methods. The two molecular techniques were significantly more sensitive than valve culture (18%) and accurately identified bacteria in excised heart valves. In eight patients with culture-negative IE, the following results were obtained: concordant detection of Coxiella burnetii (n = 2), Streptococcus gallolyticus (n = 1), Propionibacterium acnes (n = 1), and viridans group streptococci (n = 1) by both molecular tests, detection of P. acnes by PCR-ESI-MS whereas the 16S rRNA PCR was negative (n = 1), and a false-negative result by both molecular techniques (n = 2). In one case of IE caused by viridans streptococci, PCR-ESI-MS was positive for Enterococcus spp. The advantages of PCR-ESI-MS compared to 16S rRNA PCR are its automated workflow and shorter turnaround times. PMID:27629895

  13. Leaf hydraulic vulnerability to drought is linked to site water availability across a broad range of species and climates.

    PubMed

    Blackman, Chris J; Gleason, Sean M; Chang, Yvonne; Cook, Alicia M; Laws, Claire; Westoby, Mark

    2014-09-01

    Vulnerability of the leaf hydraulic pathway to water-stress-induced dysfunction is a key component of drought tolerance in plants and may be important in defining species' climatic range. However, the generality of the association between leaf hydraulic vulnerability and climate across species and sites remains to be tested. Leaf hydraulic vulnerability to drought (P50leaf, the water potential inducing 50 % loss in hydraulic function) was measured in a diverse group of 92 woody, mostly evergreen angiosperms from sites across a wide range of habitats. These new data together with some previously published were tested against key climate indices related to water availability. Differences in within-site variability in P50leaf between sites were also examined. Values of hydraulic vulnerability to drought in leaves decreased strongly (i.e. became more negative) with decreasing annual rainfall and increasing aridity across sites. The standard deviation in P50leaf values recorded within each site was positively correlated with increasing aridity. P50leaf was also a good indicator of the climatic envelope across each species' distributional range as well as their dry-end distributional limits within Australia, although this relationship was not consistently detectable within sites. The findings indicate that species sorting processes have influenced distributional patterns of P50leaf across the rainfall spectrum, but alternative strategies for dealing with water deficit exist within sites. The strong link to aridity suggests leaf hydraulic vulnerability may influence plant distributions under future climates. © The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. Construction of a self-luminescent cyanobacterial bioreporter that detects a broad range of bioavailable heavy metals in aquatic environments.

    PubMed

    Martín-Betancor, Keila; Rodea-Palomares, Ismael; Muñoz-Martín, M A; Leganés, Francisco; Fernández-Piñas, Francisca

    2015-01-01

    A self-luminescent bioreporter strain of the unicellular cyanobacterium Synechococcus sp. PCC 7942 was constructed by fusing the promoter region of the smt locus (encoding the transcriptional repressor SmtB and the metallothionein SmtA) to luxCDABE from Photorhabdus luminescens; the sensor smtB gene controlling the expression of smtA was cloned in the same vector. The bioreporter performance was tested with a range of heavy metals and was shown to respond linearly to divalent Zn, Cd, Cu, Co, Hg, and monovalent Ag. Chemical modeling was used to link bioreporter response with metal speciation and bioavailability. Limits of Detection (LODs), Maximum Permissive Concentrations (MPCs) and dynamic ranges for each metal were calculated in terms of free ion concentrations. The ranges of detection varied from 11 to 72 pM for Hg(2+) (the ion to which the bioreporter was most sensitive) to 1.54-5.35 μM for Cd(2+) with an order of decreasing sensitivity as follows: Hg(2+) > Cu(2+) > Ag(+) > Co(2+) ≥ Zn(2+) > Cd(2+). However, the maximum induction factor reached 75-fold in the case of Zn(2+) and 56-fold in the case of Cd(2+), implying that Zn(2+) is the preferred metal in vivo for the SmtB sensor, followed by Cd(2+), Ag(+) and Cu(2+) (around 45-50-fold induction), Hg(2+) (30-fold) and finally Co(2+) (20-fold). The bioreporter performance was tested in real environmental samples with different water matrix complexity artificially contaminated with increasing concentrations of Zn, Cd, Ag, and Cu, confirming its validity as a sensor of free heavy metal cations bioavailability in aquatic environments.

  15. Construction of a self-luminescent cyanobacterial bioreporter that detects a broad range of bioavailable heavy metals in aquatic environments

    PubMed Central

    Martín-Betancor, Keila; Rodea-Palomares, Ismael; Muñoz-Martín, M. A.; Leganés, Francisco; Fernández-Piñas, Francisca

    2015-01-01

    A self-luminescent bioreporter strain of the unicellular cyanobacterium Synechococcus sp. PCC 7942 was constructed by fusing the promoter region of the smt locus (encoding the transcriptional repressor SmtB and the metallothionein SmtA) to luxCDABE from Photorhabdus luminescens; the sensor smtB gene controlling the expression of smtA was cloned in the same vector. The bioreporter performance was tested with a range of heavy metals and was shown to respond linearly to divalent Zn, Cd, Cu, Co, Hg, and monovalent Ag. Chemical modeling was used to link bioreporter response with metal speciation and bioavailability. Limits of Detection (LODs), Maximum Permissive Concentrations (MPCs) and dynamic ranges for each metal were calculated in terms of free ion concentrations. The ranges of detection varied from 11 to 72 pM for Hg2+ (the ion to which the bioreporter was most sensitive) to 1.54–5.35 μM for Cd2+ with an order of decreasing sensitivity as follows: Hg2+ >> Cu2+ >> Ag+ > Co2+ ≥ Zn2+ > Cd2+. However, the maximum induction factor reached 75-fold in the case of Zn2+ and 56-fold in the case of Cd2+, implying that Zn2+ is the preferred metal in vivo for the SmtB sensor, followed by Cd2+, Ag+ and Cu2+ (around 45–50-fold induction), Hg2+ (30-fold) and finally Co2+ (20-fold). The bioreporter performance was tested in real environmental samples with different water matrix complexity artificially contaminated with increasing concentrations of Zn, Cd, Ag, and Cu, confirming its validity as a sensor of free heavy metal cations bioavailability in aquatic environments. PMID:25806029

  16. Compton scattering for spectroscopic detection of ultra-fast, high flux, broad energy range X-rays.

    PubMed

    Cipiccia, S; Wiggins, S M; Maneuski, D; Brunetti, E; Vieux, G; Yang, X; Issac, R C; Welsh, G H; Anania, M; Islam, M R; Ersfeld, B; Montgomery, R; Smith, G; Hoek, M; Hamilton, D J; Lemos, N R C; Symes, D R; Rajeev, P P; Shea, V O; Dias, J M; Jaroszynski, D A

    2013-11-01

    Compton side-scattering has been used to simultaneously downshift the energy of keV to MeV energy range photons while attenuating their flux to enable single-shot, spectrally resolved, measurements of high flux X-ray sources to be undertaken. To demonstrate the technique a 1 mm thick pixelated cadmium telluride detector has been used to measure spectra of Compton side-scattered radiation from a Cobalt-60 laboratory source and a high flux, high peak brilliance X-ray source of betatron radiation from a laser-plasma wakefield accelerator.

  17. Compton scattering for spectroscopic detection of ultra-fast, high flux, broad energy range X-rays

    SciTech Connect

    Cipiccia, S.; Wiggins, S. M.; Brunetti, E.; Vieux, G.; Yang, X.; Welsh, G. H.; Anania, M.; Islam, M. R.; Ersfeld, B.; Jaroszynski, D. A.; Maneuski, D.; Montgomery, R.; Smith, G.; Hoek, M.; Hamilton, D. J.; Shea, V. O.; Issac, R. C.; Lemos, N. R. C.; Dias, J. M.; and others

    2013-11-15

    Compton side-scattering has been used to simultaneously downshift the energy of keV to MeV energy range photons while attenuating their flux to enable single-shot, spectrally resolved, measurements of high flux X-ray sources to be undertaken. To demonstrate the technique a 1 mm thick pixelated cadmium telluride detector has been used to measure spectra of Compton side-scattered radiation from a Cobalt-60 laboratory source and a high flux, high peak brilliance X-ray source of betatron radiation from a laser-plasma wakefield accelerator.

  18. Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles.

    PubMed

    Ceuppens, Siele; Boon, Nico; Uyttendaele, Mieke

    2013-06-01

    Bacillus cereus comprises a highly versatile group of bacteria, which are of particular interest because of their capacity to cause disease. Emetic food poisoning is caused by the toxin cereulide produced during the growth of emetic B. cereus in food, while diarrhoeal food poisoning is the result of enterotoxin production by viable vegetative B. cereus cells in the small intestine, probably in the mucus layer and/or attached to the host's intestinal epithelium. The numbers of B. cereus causing disease are highly variable, depending on diverse factors linked to the host (age, diet, physiology and immunology), bacteria (cellular form, toxin genes and expression) and food (nutritional composition and meal characteristics). Bacillus cereus group strains show impressive ecological diversity, ranging from their saprophytic life cycle in soil to symbiotic (commensal and mutualistic) lifestyles near plant roots and in guts of insects and mammals to various pathogenic ones in diverse insect and mammalian hosts. During all these different ecological lifestyles, their toxins play important roles ranging from providing competitive advantages within microbial communities to inhibition of specific pathogenic organisms for their host and accomplishment of infections by damaging their host's tissues. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  19. Assessing the link between chlorophyll concentration and absorption line height at 676 nm over a broad range of water types.

    PubMed

    Nardelli, Schuyler C; Twardowski, Michael S

    2016-10-31

    The relationship between absorption at 676 nm normalized to chlorophyll-a, i.e., specific absorption aph*(676), and various optical and environmental properties is examined in extensive data sets from Case I and Case II waters found globally to assess drivers of variability such as pigment packaging. A better understanding of this variability could lead to more accurate estimates of chlorophyll concentrations from in situ optical measurements that may be made autonomously. Values of aph*(676) ranged from 0.00006 to 0.0944 m2/mg Chl a across all sites studied, but converged on median and mean values (n = 563) of 0.0108 and 0.0139 m2/mg Chl a respectively, with no apparent relationship with various optical properties, latitude, coastal or open ocean environment, depth, temperature, salinity, photoadaptation, ecosystem health, or albedo. Relative consistency in aph* across such diverse water types and the full range in chlorophyll concentration suggests a single aph* may be used to estimate chlorophyll concentration from absorption measurements with better accuracy than currently thought.

  20. Broad range analysis of endocrine disruptors and pharmaceuticals using gas chromatography and liquid chromatography tandem mass spectrometry.

    PubMed

    Trenholm, Rebecca A; Vanderford, Brett J; Holady, Janie C; Rexing, David J; Snyder, Shane A

    2006-12-01

    Endocrine disrupting compounds (EDCs) and pharmaceuticals and personal care products (PPCPs) have been globally detected in impacted natural waters. The detection of trace quantities of EDCs and PPCPs in the environment is of great concern since some of these compounds have known physiological responses at low concentrations. EDCs can have a wide range of polarities, acidic and basic moieties, and exist in trace quantities, which often requires numerous complex extractions, large sample collection volumes, and multiple instrumental analyses. A comprehensive method has been developed allowing for the analysis of 58 potential EDCs in various water matrices using a single solid-phase extraction (SPE) of a 1L sample with subsequent analyses using both gas chromatography and liquid chromatography, each coupled with tandem mass spectrometry (GC-MS/MS and LC-MS/MS). Instrument detection limits ranged between 0.12-7.5 pg with corresponding method reporting limits of 1-10 ng l(-1) in water. Recoveries for most compounds were between 50% and 112% with good reproducibility (RSD 6-22%).

  1. Broad Anatomical Variation within a Narrow Wood Density Range--A Study of Twig Wood across 69 Australian Angiosperms.

    PubMed

    Ziemińska, Kasia; Westoby, Mark; Wright, Ian J

    2015-01-01

    Just as people with the same weight can have different body builds, woods with the same wood density can have different anatomies. Here, our aim was to assess the magnitude of anatomical variation within a restricted range of wood density and explore its potential ecological implications. Twig wood of 69 angiosperm tree and shrub species was analyzed. Species were selected so that wood density varied within a relatively narrow range (0.38-0.62 g cm-3). Anatomical traits quantified included wood tissue fractions (fibres, axial parenchyma, ray parenchyma, vessels, and conduits with maximum lumen diameter below 15 μm), vessel properties, and pith area. To search for potential ecological correlates of anatomical variation the species were sampled across rainfall and temperature contrasts, and several other ecologically-relevant traits were measured (plant height, leaf area to sapwood area ratio, and modulus of elasticity). Despite the limited range in wood density, substantial anatomical variation was observed. Total parenchyma fraction varied from 0.12 to 0.66 and fibre fraction from 0.20 to 0.74, and these two traits were strongly inversely correlated (r = -0.86, P < 0.001). Parenchyma was weakly (0.24 ≤|r|≤ 0.35, P < 0.05) or not associated with vessel properties nor with height, leaf area to sapwood area ratio, and modulus of elasticity (0.24 ≤|r|≤ 0.41, P < 0.05). However, vessel traits were fairly well correlated with height and leaf area to sapwood area ratio (0.47 ≤|r|≤ 0.65, all P < 0.001). Modulus of elasticity was mainly driven by fibre wall plus vessel wall fraction rather than by the parenchyma component. Overall, there seem to be at least three axes of variation in xylem, substantially independent of each other: a wood density spectrum, a fibre-parenchyma spectrum, and a vessel area spectrum. The fibre-parenchyma spectrum does not yet have any clear or convincing ecological interpretation.

  2. Unified one-dimensional model of bounded plasma with nonzero ion temperature in a broad pressure range

    SciTech Connect

    Palacio Mizrahi, J. H.; Gurovich, V. Tz.; Krasik, Ya. E.

    2013-03-15

    A one-dimensional model for steady state plasmas bounded either between large parallel walls, or by a cylinder or a sphere, valid in a wide range of gas pressures, is considered. The model includes nonzero ion temperature, inertial terms in the ion momentum equations, and allows one to calculate the plasma electron temperature and ion current density reaching the wall, as well as the spatial distributions of the ion fluid velocity, plasma density, and plasma potential in the plasma bulk. In addition, the effect of electron inertia is analyzed. The model includes as particular cases several earlier models that were based on a similar set of differential equations, but that are restricted to a specific pressure regime (low, intermediate, or high). Analytical solution is found in planar geometry, and numerical solution is given in cylindrical and spherical geometry. The results obtained are compared with those of earlier models and the differences are analyzed.

  3. Characterization of extended range Bonner Sphere Spectrometers in the CERF high-energy broad neutron field at CERN

    NASA Astrophysics Data System (ADS)

    Agosteo, S.; Bedogni, R.; Caresana, M.; Charitonidis, N.; Chiti, M.; Esposito, A.; Ferrarini, M.; Severino, C.; Silari, M.

    2012-12-01

    The accurate determination of the ambient dose equivalent in the mixed neutron-photon fields encountered around high-energy particle accelerators still represents a challenging task. The main complexity arises from the extreme variability of the neutron energy, which spans over 10 orders of magnitude or more. Operational survey instruments, which response function attempts to mimic the fluence-to-ambient dose equivalent conversion coefficient up to GeV neutrons, are available on the market, but their response is not fully reliable over the entire energy range. Extended range rem counters (ERRC) do not require the exact knowledge of the energy distribution of the neutron field and the calibration can be done with a source spectrum. If the actual neutron field has an energy distribution different from the calibration spectrum, the measurement is affected by an added uncertainty related to the partial overlap of the fluence-to-ambient dose equivalent conversion curve and the response function. For this reason their operational use should always be preceded by an "in-field" calibration, i.e. a calibration made against a reference instrument exposed in the same field where the survey-meter will be employed. In practice the extended-range Bonner Sphere Spectrometer (ERBSS) is the only device which can serve as reference instrument in these fields, because of its wide energy range and the possibility to assess the neutron fluence and the ambient dose equivalent (H*(10)) values with the appropriate accuracy. Nevertheless, the experience gained by a number of experimental groups suggests that mandatory conditions for obtaining accurate results in workplaces are: (1) the use of a well-established response matrix, thus implying validation campaigns in reference monochromatic neutrons fields, (2) the expert and critical use of suitable unfolding codes, and (3) the performance test of the whole system (experimental set-up, elaboration and unfolding procedures) in a well

  4. Identification of endosymbionts in ticks by broad-range polymerase chain reaction and electrospray ionization mass spectrometry.

    PubMed

    Rounds, Megan A; Crowder, Christopher D; Matthews, Heather E; Philipson, Curtis A; Scoles, Glen A; Ecker, David J; Schutzer, Steven E; Eshoo, Mark W

    2012-07-01

    Many organisms, such as insects, filarial nematodes, and ticks, contain heritable bacterial endosymbionts that are often closely related to transmissible tickborne pathogens. These intracellular bacteria are sometimes unique to the host species, presumably due to isolation and genetic drift. We used a polymerase chain reaction/electrospray ionization-mass spectrometry assay designed to detect a wide range of vectorborne microorganisms to characterize endosymbiont genetic signatures from Amblyomma americanum (L.), Amblyomma maculatum Koch, Dermacentor andersoni Stiles, Dermacentor occidentalis Marx, Dermacentor variabilis (Say), Ixodes scapularis Say, Ixodes pacificus Cooley & Kohls, Ixodes ricinus (L.), and Rhipicephalus sanguineus (Latreille) ticks collected at various sites and of different stages and both sexes. The assay combines the abilities to simultaneously detect pathogens and closely related endosymbionts and to identify tick species via characterization of their respective unique endosymbionts in a single test.

  5. Identification of Endosymbionts in Ticks by Broad-Range Polymerase Chain Reaction and Electrospray Ionization Mass Spectrometry

    PubMed Central

    ROUNDS, MEGAN A.; CROWDER, CHRISTOPHER D.; MATTHEWS, HEATHER E.; PHILIPSON, CURTIS A.; SCOLES, GLEN A.; ECKER, DAVID J.; SCHUTZER, STEVEN E.; ESHOO, MARK W.

    2012-01-01

    Many organisms, such as insects, filarial nematodes, and ticks, contain heritable bacterial endosymbionts that are often closely related to transmissible tickborne pathogens. These intracellular bacteria are sometimes unique to the host species, presumably due to isolation and genetic drift. We used a polymerase chain reaction/electrospray ionization-mass spectrometry assay designed to detect a wide range of vectorborne microorganisms to characterize endosymbiont genetic signatures from Amblyomma americanum (L.), Amblyomma maculatum Koch, Dermacentor andersoni Stiles, Dermacentor occidentalis Marx, Dermacentor variabilis (Say), Ixodes scapularis Say, Ixodes pacificus Cooley & Kohls, Ixodes ricinus (L.), and Rhipicephalus sanguineus (Latreille) ticks collected at various sites and of different stages and both sexes. The assay combines the abilities to simultaneously detect pathogens and closely related endosymbionts and to identify tick species via characterization of their respective unique endosymbionts in a single test. PMID:22897044

  6. Separation and determination of chiral composition in penicillamine tablets by capillary electrophoresis in a broad pH range.

    PubMed

    Song, Lijuan; Guo, Zhenpeng; Chen, Yi

    2012-07-01

    A chiral capillary electrophoretic method with nearly full pH window was explored for the separation and determination of dl-penicillamine. A facile one-pot labeling technique was coupled in the method for introduction of chromophore and charge groups onto the analytes to facilitate the electromigration and sensitive detection. By using simply a cost-effective neutral β-cyclodextrin as chiral selector, baseline separation of the dl-penicillamine was achieved from pH 2.0 to over pH 10. Quantification of standard d- and l-penicillamines was demonstrated by taking pH 4.5, 7.4, and 9.7 as the representatives of acidic, neutral, and basic conditions. The working curves were constructed between peak area and concentration, having linear ranges of 8.56-8.56 × 10(2) μg/mL for pH 4.5 and 8.56-1.71 × 10(3) μg/mL for pH 7.4 and 9.7, with correlation coefficients all better than 0.999. The limit of detection (S/N = 3) was 2.58 μg/mL in acidic and neutral conditions or 1.41 μg/mL in basic condition. The method was further validated by assaying the commercial penicillamine tablets, applicable to quantification of the effective enantiomer and the trace impurity of l-penicillamine at a content of down to 0.2, 0.6, and 2.0% for pH 9.7, 4.5, and 7.4, respectively. The recovery determined by spiking technique was in a range from 93.1 to 105 %. The method is easily extendable to the analysis of other chiral amines or amino acids.

  7. Myxobacteria Are Able to Prey Broadly upon Clinically-Relevant Pathogens, Exhibiting a Prey Range Which Cannot Be Explained by Phylogeny

    PubMed Central

    Livingstone, Paul G.; Morphew, Russell M.; Whitworth, David E.

    2017-01-01

    Myxobacteria are natural predators of microorganisms and the subjects of concerted efforts to identify novel antimicrobial compounds. Myxobacterial predatory activity seems to require more than just the possession of specific antimicrobial metabolites. Thus a holistic approach to studying predation promises novel insights into antimicrobial action. Here, we report the isolation of 113 myxobacteria from samples of soil taken from a range of habitats in mid Wales. Predatory activity of each isolate was quantified against a panel of clinically important prey organisms, including Klebsiella pneumoniae, Proteus mirabilis, Candida albicans, Enterococcus faecalis, and three species of Staphylococcus. Myxobacterial isolates exhibited a wide range of predation activity profiles against the panel of prey. Efficient predation of all prey by isolates within the collection was observed, with K. pneumoniae and C. albicans proving particularly susceptible to myxobacterial predation. Notably efficient predators tended to be proficient at predating multiple prey organisms, suggesting they possess gene(s) encoding a broad range killing activity. However, predatory activity was not congruent with phylogeny, suggesting prey range is subject to relatively rapid specialization, potentially involving lateral gene transfer. The broad but patchy prey ranges observed for natural myxobacterial isolates also implies multiple (potentially overlapping) genetic determinants are responsible for dictating predatory activity.

  8. Myxobacteria Are Able to Prey Broadly upon Clinically-Relevant Pathogens, Exhibiting a Prey Range Which Cannot Be Explained by Phylogeny.

    PubMed

    Livingstone, Paul G; Morphew, Russell M; Whitworth, David E

    2017-01-01

    Myxobacteria are natural predators of microorganisms and the subjects of concerted efforts to identify novel antimicrobial compounds. Myxobacterial predatory activity seems to require more than just the possession of specific antimicrobial metabolites. Thus a holistic approach to studying predation promises novel insights into antimicrobial action. Here, we report the isolation of 113 myxobacteria from samples of soil taken from a range of habitats in mid Wales. Predatory activity of each isolate was quantified against a panel of clinically important prey organisms, including Klebsiella pneumoniae, Proteus mirabilis, Candida albicans, Enterococcus faecalis, and three species of Staphylococcus. Myxobacterial isolates exhibited a wide range of predation activity profiles against the panel of prey. Efficient predation of all prey by isolates within the collection was observed, with K. pneumoniae and C. albicans proving particularly susceptible to myxobacterial predation. Notably efficient predators tended to be proficient at predating multiple prey organisms, suggesting they possess gene(s) encoding a broad range killing activity. However, predatory activity was not congruent with phylogeny, suggesting prey range is subject to relatively rapid specialization, potentially involving lateral gene transfer. The broad but patchy prey ranges observed for natural myxobacterial isolates also implies multiple (potentially overlapping) genetic determinants are responsible for dictating predatory activity.

  9. Preanalytic removal of human DNA eliminates false signals in general 16S rDNA PCR monitoring of bacterial pathogens in blood.

    PubMed

    Handschur, Michael; Karlic, Heidrun; Hertel, Christian; Pfeilstöcker, Michael; Haslberger, Alexander G

    2009-05-01

    PCR detection of microbial pathogens in blood from patients is a promising issue for rapid diagnosis of sepsis and early targeted therapy. However, for PCR assays detecting all bacterial groups, broad range primers, in particular the 16S rDNA targeting primers have to be used. Upcoming false signals and reduced sensitivity are a common problem as a consequence of unspecific amplification reactions with the human DNA background. Here we show that, using total DNA extracts from blood, unspecific signals occurred in general 16S rDNA PCRs as a result of the amplification of human sequences. To address this problem, we developed a protocol by which the human background DNA is removed and bacterial DNA is enriched during sample preparation, a method we termed background-free enrichment method (BFEM). In general, we aimed to exclude false signals due to the human background DNA yielded from 16S rDNA PCR, Real-Time-PCR and IGS-PCR analyses. We applied the BFEM to the analysis of blood samples from 22 patients and obtained results similar to standard blood culture methods. The BFEM allows specific and sensitive detection of pathogens in downstream PCR assays and is easy to handle due to the quick sample preparation procedure. Thus, the BFEM contributes to the generation of replicable and more reliable data in general 16S rDNA PCR assays.

  10. A Multivalent Adsorption Apparatus Explains the Broad Host Range of Phage phi92: a Comprehensive Genomic and Structural Analysis

    PubMed Central

    Buettner, Falk F. R.; Browning, Christopher; Nazarov, Sergey; Rabsch, Wolfgang; Bethe, Andrea; Oberbeck, Astrid; Bowman, Valorie D.; Stummeyer, Katharina; Mühlenhoff, Martina; Gerardy-Schahn, Rita

    2012-01-01

    Bacteriophage phi92 is a large, lytic myovirus isolated in 1983 from pathogenic Escherichia coli strains that carry a polysialic acid capsule. Here we report the genome organization of phi92, the cryoelectron microscopy reconstruction of its virion, and the reinvestigation of its host specificity. The genome consists of a linear, double-stranded 148,612-bp DNA sequence containing 248 potential open reading frames and 11 putative tRNA genes. Orthologs were found for 130 of the predicted proteins. Most of the virion proteins showed significant sequence similarities to proteins of myoviruses rv5 and PVP-SE1, indicating that phi92 is a new member of the novel genus of rv5-like phages. Reinvestigation of phi92 host specificity showed that the host range is not limited to polysialic acid-encapsulated Escherichia coli but includes most laboratory strains of Escherichia coli and many Salmonella strains. Structure analysis of the phi92 virion demonstrated the presence of four different types of tail fibers and/or tailspikes, which enable the phage to use attachment sites on encapsulated and nonencapsulated bacteria. With this report, we provide the first detailed description of a multivalent, multispecies phage armed with a host cell adsorption apparatus resembling a nanosized Swiss army knife. The genome, structure, and, in particular, the organization of the baseplate of phi92 demonstrate how a bacteriophage can evolve into a multi-pathogen-killing agent. PMID:22787233

  11. Exploration of Daphnia behavioral effect profiles induced by a broad range of toxicants with different modes of action.

    PubMed

    Chevalier, Julie; Harscoët, Elodie; Keller, Merlin; Pandard, Pascal; Cachot, Jérôme; Grote, Matthias

    2015-08-01

    Behavior is increasingly reported as a sensitive and early indicator of toxicant stress in aquatic organisms. However, the systematic understanding of behavioral effects and comparisons between effect profiles is hampered because the available studies are limited to few chemicals and differ in the exposure conditions and effect parameters examined. The aims of the present study were 1) to explore behavioral responses of Daphnia magna exposed to different toxicants, 2) to compare behavioral effect profiles with regard to chemical modes of action, and 3) to determine the sensitivity and response time of behavioral parameters in a new multi-cell exposure system named Multi-DaphTrack compared with currently utilized tests. Twelve compounds covering different modes of toxic action were selected to sample a wide range of potential effect profiles. Acute standard immobilization tests and 48 h of behavioral tracking were performed in the customized Multi-DaphTrack system and a single-cell commercialized biological early warning system. Contrasting behavioral profiles were observed for average speed (i.e., intensity, time of effect onset, effect duration), but no distinct behavioral profiles could be drawn from the chemical mode of action. Most compounds tested in the Multi-DaphTrack system induced an early and significant average speed increase at concentrations near or below the 10% effective concentration (48 h) of the acute immobilization test, demonstrating that the Multi-DaphTrack system is fast and sensitive. To conclude, behavior endpoints could be used as an alternative or complement to the current acute standard test or chemical analysis for the predictive evaluation of ecotoxic effects of effluents or water bodies. © 2015 SETAC.

  12. Two Measurement Methods of Leaf Dry Matter Content Produce Similar Results in a Broad Range of Species

    PubMed Central

    Vaieretti, María Victoria; Díaz, Sandra; Vile, Denis; Garnier, Eric

    2007-01-01

    Background and Aims Leaf dry matter content (LDMC) is widely used as an indicator of plant resource use in plant functional trait databases. Two main methods have been proposed to measure LDMC, which basically differ in the rehydration procedure to which leaves are subjected after harvesting. These are the ‘complete rehydration’ protocol of Garnier et al. (2001, Functional Ecology 15: 688–695) and the ‘partial rehydration’ protocol of Vendramini et al. (2002, New Phytologist 154: 147–157). Methods To test differences in LDMC due to the use of different methods, LDMC was measured on 51 native and cultivated species representing a wide range of plant families and growth forms from central-western Argentina, following the complete rehydration and partial rehydration protocols. Key Results and Conclusions The LDMC values obtained by both methods were strongly and positively correlated, clearly showing that LDMC is highly conserved between the two procedures. These trends were not altered by the exclusion of plants with non-laminar leaves. Although the complete rehydration method is the safest to measure LDMC, the partial rehydration procedure produces similar results and is faster. It therefore appears as an acceptable option for those situations in which the complete rehydration method cannot be applied. Two notes of caution are given for cases in which different datasets are compared or combined: (1) the discrepancy between the two rehydration protocols is greatest in the case of high-LDMC (succulent or tender) leaves; (2) the results suggest that, when comparing many studies across unrelated datasets, differences in the measurement protocol may be less important than differences among seasons, years and the quality of local habitats. PMID:17353207

  13. Estimated Glomerular Filtration Rate Is a Poor Predictor of Concentration for a Broad Range of Uremic Toxins

    PubMed Central

    Schepers, Eva; Barreto, Daniela V.; Barreto, Fellype C.; Liabeuf, Sophie; Van Biesen, Wim; Verbeke, Francis; Glorieux, Griet; Choukroun, Gabriel; Massy, Ziad; Vanholder, Raymond

    2011-01-01

    Summary Background and objectives The degree of chronic kidney disease (CKD) is currently expressed in terms of GFR, which can be determined directly or estimated according to different formulas on the basis of serum creatinine and/or cystatin C measurements (estimated GFR [eGFR]). The purpose of this study was to investigate whether eGFR values are representative for uremic toxin concentrations in patients with different degrees of CKD. Design, setting, participants, & measurements Associations between eGFR based on serum cystatin C and different uremic solutes (mol wt range 113 to 240 D; determined by colorimetry, HPLC, or ELISA) were evaluated in 95 CKD patients not on dialysis (CKD stage 2 to 5). The same analysis was also applied for six other eGFR formulas. Results There was a substantial disparity in fits among solutes. In linear regression, explained variance of eGFR was extremely low for most solutes, with eGFR > 0.4 only for creatinine. The other eGFR formulations gave comparably disappointing results with regard to their association to uremic solutes. Relative similarity in R2 values per solute for the different eGFR values and the strong disparity in values between solutes suggest that the differences in R2 are mainly due to discrepancies in solute handling apart from GFR. Conclusions eGFR is poorly associated with concentrations of all studied uremic toxins in patients with different degrees of CKD, correlates differently with each individual solute, and can thus not be considered representative for evaluating the accumulation of solutes in the course of CKD. PMID:21617084

  14. [Characterization of mid-subtropical evergreen broad-leaved forest gap based on light detection and ranging (LiDAR)].

    PubMed

    Liu, Feng; Tan, Chang; Wang, Hong; Zhang, Jiang; Wan, Ying; Long, Jiang-ping; Liu, Rui-xi

    2015-12-01

    Light Detection and Ranging (LiDAR) is an active remote sensing technology for acqui- ring three-dimensional structure parameters of vegetation canopy with high accuracy over multiple spatial scales, which is greatly important to the promotion of forest disturbance ecology and the ap- plication on gaps. This paper focused on mid-subtropical evergreen broadleaved forest in Hunan Province, and small footprint LiDAR point data were adopted to identify canopy gaps. and measure geomagnetic characteristics of gaps. The optimal grid model resolution and interpolation methods were chosen to generate canopy height model, and the computer graphics processing was adopted to estimate characteristics of gaps which involved gap size, canopy height and gap shape index, then field investigation was utilized to validate the estimation results. The results showed that the gap rec- ognition rate was 94.8%, and the major influencing factors were gap size and gap maker type. Line- ar correlation was observed between LiDAR estimation and field investigation, and the R² values of gap size and canopy height case were 0.962 and 0.878, respectively. Compared with field investiga- tion, the size of mean estimated gap was 19.9% larger and the mean estimated canopy height was 9.9% less. Gap density was 12.8 gaps · hm⁻² and the area of gaps occupied 13.3% of the forest area. The average gap size, canopy height and gap shape index were 85.06 m², 15.33 m and 1.71, respectively. The study site usually contained small gaps in which the edge effect was not obvious.

  15. Light acclimation maintains the redox state of the PS II electron acceptor Q(A) within a narrow range over a broad range of light intensities.

    PubMed

    Rosenqvist, E

    2001-01-01

    Chrysanthemum inducum-hybrid 'Coral Charm', Hibiscus rosa-sinensis L. 'Cairo Red' and Spathiphyllum wallisii Regel 'Petit' were grown in natural light in a greenhouse at three levels of irradiance using permanent shade screens. Light acclimation of photosynthesis was characterized using modulated chlorophyll a fluorescence of intact leaves. A close correlation was found between the degree of reduction of the primary electron acceptor Q(A) of Photosystem II (PS II) approximated as the fluorescence parameter 1-q(P), and light acclimation. The action range of 1-q(P) was 0-0.4 from darkness to full irradiance around noon, within the respective light treatments in the greenhouse, indicating that most PS II reaction centres were kept open. In general, the index for electron transport (ETR) measured by chlorophyll fluorescence was higher for high-light (HL) than intermediate-(IL) and low-light (LL) grown plants. However, HL Chrysanthemum showed 40% higher ETR than HL Hibiscus at light saturation, despite identical redox states of Q(A). The light acclimation of the non-radiative dissipation of excess energy in the antenna, NPQ, varied considerably between the species. However, when normalized against q(P), a strong negative correlation was found between thermal dissipation and ETR measured by chlorophyll fluorescence. To be able to accommodate a high flux of electrons through PS II, the plants with the highest light-saturated ETR had the lowest NPQ/q(P). The possibility of using chlorophyll fluorescence for quantification of the energy balance between energy input and utilization in PS II in intact leaves is discussed.

  16. Diversity of thermophiles in a Malaysian hot spring determined using 16S rRNA and shotgun metagenome sequencing

    PubMed Central

    Chan, Chia Sing; Chan, Kok-Gan; Tay, Yea-Ling; Chua, Yi-Heng; Goh, Kian Mau

    2015-01-01

    The Sungai Klah (SK) hot spring is the second hottest geothermal spring in Malaysia. This hot spring is a shallow, 150-m-long, fast-flowing stream, with temperatures varying from 50 to 110°C and a pH range of 7.0–9.0. Hidden within a wooded area, the SK hot spring is continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC). In this study, a sample taken from the middle of the stream was analyzed at the 16S rRNA V3-V4 region by amplicon metagenome sequencing. Over 35 phyla were detected by analyzing the 16S rRNA data. Firmicutes and Proteobacteria represented approximately 57% of the microbiome. Approximately 70% of the detected thermophiles were strict anaerobes; however, Hydrogenobacter spp., obligate chemolithotrophic thermophiles, represented one of the major taxa. Several thermophilic photosynthetic microorganisms and acidothermophiles were also detected. Most of the phyla identified by 16S rRNA were also found using the shotgun metagenome approaches. The carbon, sulfur, and nitrogen metabolism within the SK hot spring community were evaluated by shotgun metagenome sequencing, and the data revealed diversity in terms of metabolic activity and dynamics. This hot spring has a rich diversified phylogenetic community partly due to its natural environment (plant litter, high TOC, and a shallow stream) and geochemical parameters (broad temperature and pH range). It is speculated that symbiotic relationships occur between the members of the community. PMID:25798135

  17. Laboratory study on the kinetics of CO2 hydrates in a broad p-T range relevant to Mars

    NASA Astrophysics Data System (ADS)

    Falenty, A.; Kuhs, W. F.

    2007-08-01

    , temporary gas outbursts are conceivable. Between 190K and 240K neither annealing of defective ice Ih nor the crystal regrowth is fast enough to effectively slow down outward diffusing gas molecules. In such a scenario slow decomposition is to be expected and therefore the impact on the surface will be very limited. Surprisingly we also have found "self preservation" in a narrow pressure range. The sealing effect is, however, less pronounced as the preservation mechanism differs from the higher temperature one. Therefore only large agglomerations of CO2 hydrates may be effectively saved from further decomposition. [1] J.S.Kargel Mars: A Warmer Wetter Planet, Springer Berlin, 2004. [2] R. Greve, R.A. Mahajan (2005), Icarus 176, 475-485 [3] D.K. Staykova et al. (2003) J. Phys. Chem. B 107,10299-10311 [4] G. Genov et al. (2004), Am. Miner. 89, 1228-1239 [5] W.F. Kuhs et al. (2006) J.Phys.Chem. B 110 (26), 13283-13295 [6] G. Genov PhD thesis, Georg-August Universität, Göttingen, 2004 [7] W. F. Kuhs et al. (2004), Phys. Chem. Chem. Phys. 6, 4917-4920 [8] A. Falenty et al. (2007) In: Physics and Chemistry of Ice (ed. W. F. Kuhs), RSC Publishing, Cambridge, 2007, pp. 171-179

  18. Hierarchical MoS2@MoP core-shell heterojunction electrocatalysts for efficient hydrogen evolution reaction over a broad pH range

    NASA Astrophysics Data System (ADS)

    Wu, Aiping; Tian, Chungui; Yan, Haijing; Jiao, Yanqing; Yan, Qing; Yang, Guoyu; Fu, Honggang

    2016-05-01

    A low-cost catalyst for the hydrogen evolution reaction (HER) over a broad pH range is highly desired to meet the practical needs in different areas. In this study, hierarchical flower-like MoS2@MoP core-shell heterojunctions (HF-MoSP) are designed as a promising catalyst for HER over a broad pH range. The materials are obtained by the controllable phosphidation of the hierarchical MoS2 flower (HF-MoS2) composed of thin silk belt-like sheets. The phosphidation degree, P/S ratio and work function (WF) of HF-MoSP can be tuned easily over broad range by changing the phosphidation temperature. Under optimized condition, HF-MoSP exhibits excellent electrocatalytic activity for HER with a low onset overpotential of 29 mV and η of 108 mV at 10 mA cm-2 in 0.5 M H2SO4 and retains its good activity for 30 h. In addition, the catalyst shows excellent activity in 1 M KOH with an onset overpotential of 42 mV and η of 119 mV at 10 mA cm-2. The catalysts also exhibit obvious activity in neutral, weak acid and weak alkaline conditions. The good performance is relative to the synergy of the MoP shell and MoS2 core and the high WF of HF-MoSP close to Pt, and the large SBET of HF-MoSP benefited from the hierarchical structure. This study represents the construction of the core-shell heterojunction and provides a new way to provide the low-cost and high-performance catalyst for HER.A low-cost catalyst for the hydrogen evolution reaction (HER) over a broad pH range is highly desired to meet the practical needs in different areas. In this study, hierarchical flower-like MoS2@MoP core-shell heterojunctions (HF-MoSP) are designed as a promising catalyst for HER over a broad pH range. The materials are obtained by the controllable phosphidation of the hierarchical MoS2 flower (HF-MoS2) composed of thin silk belt-like sheets. The phosphidation degree, P/S ratio and work function (WF) of HF-MoSP can be tuned easily over broad range by changing the phosphidation temperature. Under optimized

  19. Linkage disequilibrium mapping places the gene causing familial Mediterranean fever close to D16S246

    SciTech Connect

    Levy, E. N.; Aksentijevich, I.; Pras, E.

    1996-03-01

    This report presents refined genetic mapping data for the gene causing familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation. We sampled 65 Jewish, Armenian, and Arab families and typed them for eight markers from chromosome 16p. Using a new algorithm that permits multipoint calculations for a dense map of markers in consanguineous families, we obtained a maximal LOD score of 49.2 at a location 1.6 cM centromeric to D16S246. A specific haplotype at D16S283-D16S94-D16S246 was found in 76% of Moroccan and 32% of non-Moroccan Jewish carrier chromosomes, but this haplotype was not overrepresented in Armenian or Arab FMF carriers. Moreover, the 2.5-kb allele at D16S246 was significantly associated with FMF in Moroccan and non-Moroccan Jews but not in Armenians or Arabs. Since the Moroccan Jewish community represents a relatively recently established and genetically isolated founder population, we analyzed the Moroccan linkage-disequilibrium data by using Luria-Delbruck formulas and simulations based on a Poisson branching process. These methods place the FMF susceptibility gene within 0.305 cM of D16S246 (2-LOD-unit range 0.02-0.64 cM). 41 refs., 3 figs., 5 tabs.

  20. Genomic and functional characterization of the modular broad-host-range RA3 plasmid, the archetype of the IncU group.

    PubMed

    Kulinska, Anna; Czeredys, Magdalena; Hayes, Finbarr; Jagura-Burdzy, Grazyna

    2008-07-01

    IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha- and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta- and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids.

  1. Fluorescence ratiometric optical broad range pH sensor based on CdSe/ZnS quantum dots and O170 embedded in ethyl cellulose matrix

    NASA Astrophysics Data System (ADS)

    Chu, Cheng-Shane; Su, Chih-Jen

    2017-04-01

    This paper presents a ratiometric optical broad range pH sensor that comprises an optical fiber coated at one end with CdSe/ZnS quantum dots (QDs) and Oxazine 170 perchlorate (O170) embedded in ethyl cellulose (EC) matrix. The feasibility of coating an optical fiber with the sensing film to fabricate a ratiometric fiber optic broad range pH sensor is investigated. Using an LED with a central wavelength 405 nm as an excitation light source, the CdSe/ZnS QDs shows that the emission wavelength at 575 nm highly sensitive to pH and O170 shows the emission wavelength at 655 nm insensitive to pH. The ratio of fluorescence intensities at 575 nm and 655 nm has a linear relationship with pH in the 0.9-12.2 range. The ratiometric sensing approach presented in this study has the advantage of suppressing spurious fluctuations in the intensity of the excitation source and optical transmission properties of the optical fiber sensor.

  2. Ro-vibrational quenching of CO (v = 1) by He impact in a broad range of temperatures: A benchmark study using mixed quantum/classical inelastic scattering theory.

    PubMed

    Semenov, Alexander; Ivanov, Mikhail; Babikov, Dmitri

    2013-08-21

    The mixed quantum/classical approach is applied to the problem of ro-vibrational energy transfer in the inelastic collisions of CO(v = 1) with He atom, in order to predict the quenching rate coefficient in a broad range of temperatures 5 < T < 2500 K. Scattering calculations are done in two different ways: direct calculations of quenching cross sections and, alternatively, calculations of the excitation cross sections plus microscopic reversibility. In addition, a symmetrized average-velocity method of Billing is tried. Combination of these methods allows reproducing experiment in a broad range of temperatures. Excellent agreement with experiment is obtained at 400 < T < 2500 K (within 10%), good agreement in the range 100 < T < 400 K (within 25%), and semi-quantitative agreement at 40 < T < 100 K(within a factor of 2). This study provides a stringent test of the mixed quantum/classical theory, because the vibrational quantum in CO molecule is rather large and the quencher is very light (He atom). For heavier quenchers and closer to dissociation limit of the molecule, the mixed quantum/classical theory is expected to work even better.

  3. Photodetectors: Broad Detection Range Rhenium Diselenide Photodetector Enhanced by (3-Aminopropyl)Triethoxysilane and Triphenylphosphine Treatment (Adv. Mater. 31/2016).

    PubMed

    Jo, Seo-Hyeon; Park, Hyung-Youl; Kang, Dong-Ho; Shim, Jaewoo; Jeon, Jaeho; Choi, Seunghyuk; Kim, Minwoo; Park, Yongkook; Lee, Jaehyeong; Song, Young Jae; Lee, Sungjoo; Park, Jin-Hong

    2016-08-01

    The effects of triphenylphosphine (PPh3 ) and (3-amino-propyl)triethoxysilane (APTES) on a rhenium diselenide (ReSe2 ) photodetector are systematically studied by J.-H. Park and co-workers on page 6711 in comparison with a conventional MoS2 device. A very high performance ReSe2 photodetector is demonstrated, which has a broad photodetection range, high photoresponsivity (1.18 × 10(6) A W(-1) ), and fast photoswitching speed (rising/decaying time: 58/263 ms). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. High-density universal 16S rRNA microarray analysis revealsbroader diversity than typical clone library when sampling theenvironment

    SciTech Connect

    DeSantis, Todd Z.; Brodie, Eoin L.; Moberg, Jordan P.; Zubieta,Ingrid X.; Piceno, Yvette M.; Andersen, Gary L.

    2006-06-15

    Molecular approaches aimed at detection of a broad-range ofprokaryotes in the environment routinely rely upon classifyingheterogeneous 16S rRNA genes amplified by PCR using primers with broadspecificity. The general method of sampling and categorizing DNA has beento clone then sequence the PCR products. However, the number of clonesrequired to adequately catalogue the majority of taxa in a sample isunwieldy. Alternatively, hybridizing target sequences to a universal 16SrRNA gene microarray may provide a more rapid and comprehensive view ofprokaryotic community composition. This study investigated the breadthand accuracy of a microarray in detecting diverse 16S rRNA gene sequencetypes compared to clone-and-sequencing using three environmental samples:urban aerosol, subsurface soil and subsurface water. PCR productsgenerated from universal 16S rRNA gene-targeted primers were classifiedusing either the clone-and-sequence method or by hybridization to a novelhigh-density microarray of 297,851 probes complementary to 842prokaryotic sub-families. The three clone libraries comprised 1,391high-quality sequences. Approximately 8 percent of the clones could notbe placed into a known sub-family and were considered novel. Themicroarray results confirmed the majority of clone-detected sub-familiesand additionally demonstrated greater amplicon diversity extending intophyla not observed by the cloning method. Sequences matching OTUs withinthe phyla Nitrospira, Planctomycetes, and TM7, which were uniquelydetected by the array, were verified with specific primers and subsequentamplicon sequencing. Sub-family richness detected by the arraycorresponded well with non-parametric richness predictions extrapolatedfrom clone libraries except in the water community where clone-basedrichness predictions were greatly exceeded. It was concluded thatalthough the microarray is unreliable inidentifying novel prokaryotictaxa, it reveals greater diversity in environmental samples thansequencing a

  5. DNRA and Denitrification Coexist over a Broad Range of Acetate/N-NO3− Ratios, in a Chemostat Enrichment Culture

    PubMed Central

    van den Berg, Eveline M.; Boleij, Marissa; Kuenen, J. Gijs; Kleerebezem, Robbert; van Loosdrecht, Mark C. M.

    2016-01-01

    Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) compete for nitrate in natural and engineered environments. A known important factor in this microbial competition is the ratio of available electron donor and elector acceptor, here expressed as Ac/N ratio (acetate/nitrate-nitrogen). We studied the impact of the Ac/N ratio on the nitrate reduction pathways in chemostat enrichment cultures, grown on acetate mineral medium. Stepwise, conditions were changed from nitrate limitation to nitrate excess in the system by applying a variable Ac/N ratio in the feed. We observed a clear correlation between Ac/N ratio and DNRA activity and the DNRA population in our reactor. The DNRA bacteria dominated under nitrate limiting conditions in the reactor and were outcompeted by denitrifiers under limitation of acetate. Interestingly, in a broad range of Ac/N ratios a dual limitation of acetate and nitrate occurred with co-occurrence of DNRA bacteria and denitrifiers. To explain these observations, the system was described using a kinetic model. The model illustrates that the Ac/N effect and concomitant broad dual limitation range related to the difference in stoichiometry between both processes, as well as the differences in electron donor and acceptor affinities. Population analysis showed that the presumed DRNA-performing bacteria were the same under nitrate limitation and under dual limiting conditions, whereas the presumed denitrifying population changed under single and dual limitation conditions. PMID:27933040

  6. The Staphylococcus aureus leucine aminopeptidase LAP is localized to the bacterial cytosol and demonstrates a broad substrate range that extends beyond leucine

    PubMed Central

    Carroll, Ronan K.; Veillard, Florian; Gagne, Danielle T.; Lindenmuth, Jarrod M.; Poreba, Marcin; Drag, Marcin; Potempa, Jan; Shaw, Lindsey N.

    2013-01-01

    Staphylococcus aureus is a potent pathogen of humans exhibiting a broad disease range, in part, due to an extensive repertoire of secreted virulence factors, including proteases. Recently, we identified the first example of an intracellular protease (leucine aminopeptidase - LAP) that is required for virulence in S. aureus. Disruption of pepZ, the gene encoding LAP, had no affect on the growth rate of bacteria, however, in systemic and localized infection models the pepZ mutant was significantly attenuated in virulence. Recently, a contradictory report has been published, suggesting that LAP is an extracellular enzyme and it is required for growth in S. aureus. Here, we investigate these results and confirm our previous findings that LAP is localized to the bacterial cytosol and is not required for growth. In addition we conduct a biochemical investigation of purified recombinant LAP identifying optimal conditions for enzymatic activity and substrate preference for hydrolysis. Our results show that LAP has a broad substrate range, including activity against the dipeptide cysteine-glycine and that leucine is not the primary target of LAP. PMID:23241672

  7. The Staphylococcus aureus leucine aminopeptidase is localized to the bacterial cytosol and demonstrates a broad substrate range that extends beyond leucine.

    PubMed

    Carroll, Ronan K; Veillard, Florian; Gagne, Danielle T; Lindenmuth, Jarrod M; Poreba, Marcin; Drag, Marcin; Potempa, Jan; Shaw, Lindsey N

    2013-06-01

    Staphylococcus aureus is a potent pathogen of humans exhibiting a broad disease range, in part due to an extensive repertoire of secreted virulence factors, including proteases. Recently, we identified the first example of an intracellular protease (leucine aminopeptidase, LAP) that is required for virulence in S. aureus. Disruption of pepZ, the gene encoding LAP, had no affect on the growth rate of bacteria; however, in systemic and localized infection models the pepZ mutant had significantly attenuated virulence. Recently, a contradictory report was published suggesting that LAP is an extracellular enzyme and it is required for growth in S. aureus. Here, we investigate these results and confirm our previous findings that LAP is localized to the bacterial cytosol and is not required for growth. In addition, we conduct a biochemical investigation of purified recombinant LAP, identifying optimal conditions for enzymatic activity and substrate preference for hydrolysis. Our results show that LAP has a broad substrate range, including activity against the dipeptide cysteine-glycine, and that leucine is not the primary target of LAP.

  8. Broad-range TRP channel inhibitors (2-APB, flufenamic acid, SKF-96365) affect differently contraction of resistance and conduit femoral arteries of rat.

    PubMed

    Bencze, Michal; Behuliak, Michal; Vavřínová, Anna; Zicha, Josef

    2015-10-15

    Transient receptor potential (TRP) channels are proposed to contribute to membrane depolarization and Ca2+ influx into vascular smooth muscle (VSM) cells. Our aim was to study the effects of widely used broad-range TRP channel inhibitors--2-aminoethoxydiphenyl borate (2-APB), flufenamic acid (FFA) and SKF-96365--on the contraction of freshly isolated small and large arteries. Endothelium-denuded resistance (≈250 µm) and conduit (≈1000 µm) femoral arteries were isolated from adult Wistar rats and mounted in wire myograph. The effects of the above mentioned TRP channel inhibitors and voltage-dependent calcium channel inhibitor nifedipine were studied on arterial contractions induced by phenylephrine, U-46619 or K+. Phenylephrine-induced contractions were also studied in the absence of extracellular Na+. mRNA expression of particular canonical and melastatin TRP channel subunits in femoral vascular bed was determined. TRP channel inhibitors attenuated K+-induced contraction less than nifedipine. Phenylephrine-induced contraction was more influenced by 2-APB in resistance arteries, while FFA completely prevented U-46619-induced contraction in both sizes of arteries. The absence of extracellular Na+ prevented the inhibitory effects of 2-APB, but not those of FFA. The observed effects of broad-range TRP channel inhibitors, which were dependent on the size of the artery, confirmed the involvement of TRP channels in agonist-induced contractions. The inhibitory effects of 2-APB (but not those of FFA or SKF-96365) were dependent on the presence of extracellular Na+.

  9. Novel genus-specific broad range primers for the detection of furoviruses, hordeiviruses and rymoviruses and their application in field surveys in South-East Australia.

    PubMed

    Zheng, Linda; Tang, Joe; Clover, Gerard R G; Spackman, Merrin E; Freeman, Angela J; Rodoni, Brendan C

    2015-03-01

    A number of viruses from the genera Furovirus, Hordeivirus and Rymovirus are known to infect and damage the four major temperate cereal crops, wheat, barley, sorghum and oats. Currently, there is no active testing in Australia for any of these viruses, which pose a significant biosecurity threat to the phytosanitary status of Australia's grains industry. To address this, broad spectrum PCR assays were developed to target virus species within the genera Furovirus, Hordeivirus and Rymovirus. Five sets of novel genus-specific primers were designed and tested in reverse-transcription polymerase chain reaction assays against a range of virus isolates in plant virus diagnostic laboratories in both Australia and New Zealand. Three of these assays were then chosen to screen samples in a three-year survey of cereal crops in western Victoria, Australia. Of the 8900 cereal plants screened in the survey, all were tested free of furoviruses, hordeiviruses and rymoviruses. To date, there were no published genus-specific primers available for the detection of furoviruses, hordeiviruses and rymoviruses. This study shows for the first time a broad-spectrum molecular test being used in a survey for exotic grain viruses in Australia. Results from this survey provide important evidence of the use of this method to demonstrate the absence of these viruses in Victoria, Australia. The primer pairs reported here are expected to detect a wide range of virus species within the three genera.

  10. Effect of hydrostatic pressure on the conductivity of YBa2Cu3O7-δsingle crystals in a broad range of temperature and oxygen content

    NASA Astrophysics Data System (ADS)

    Vovk, R. V.; Khadzhai, G. Ya.; Dobrovolskiy, O. V.; Nazyrov, Z. F.; Kamchatnaya, S. N.

    2017-04-01

    The effect of external hydrostatic pressure on the electrical resistance of optimally doped and underdoped YBa2Cu3O7-δ single crystals is investigated in a broad temperature range, which includes the normal state and the region of superconducting fluctuations. The temperature dependences of the resistivity in the normal state are determined by scattering of charge carriers by phonons and defects. The application of pressure leads to significant changes in the electronic structure of the sample and reduces the degree of their defectiveness. This is accompanied by changeû in the lattice characteristics as the sample volume decreases. The fluctuation conductivity only exists within the range of ∼ 0.1Tc . With increasing δ the superconducting characteristics are close to the values typical for conventional low-temperature superconductors.

  11. Analysis of 16S rRNA gene sequences and circulating cell-free DNA from plasma of chronic fatigue syndrome and non-fatigued subjects

    PubMed Central

    Vernon, Suzanne D; Shukla, Sanjay K; Conradt, Jennifer; Unger, Elizabeth R; Reeves, William C

    2002-01-01

    Background The association of an infectious agent with chronic fatigue syndrome (CFS) has been difficult and is further complicated by the lack of a known lesion or diseased tissue. Cell-free plasma DNA could serve as a sentinel of infection and disease occurring throughout the body. This type of systemic sample coupled with broad-range amplification of bacterial sequences was used to determine whether a bacterial pathogen was associated with CFS. Plasma DNA from 34 CFS and 55 non-fatigued subjects was assessed to determine plasma DNA concentration and the presence of bacterial 16S ribosomal DNA (rDNA) sequences. Results DNA was isolated from 81 (91%) of 89 plasma samples. The 55 non-fatigued subjects had higher plasma DNA concentrations than those with CFS (average 151 versus 91 ng) and more CFS subjects (6/34, 18%) had no detectable plasma DNA than non-fatigued subjects (2/55, 4%), but these differences were not significant. Bacterial sequences were detected in 23 (26%) of 89. Only 4 (14%) CFS subjects had 16S rDNA sequences amplified from plasma compared with 17 (32%) of the non-fatigued (P = 0.03). All but 1 of the 23 16S rDNA amplicon-positive subjects had five or more unique sequences present. Conclusions CFS subjects had slightly lower concentrations or no detectable plasma DNA than non-fatigued subjects. There was a diverse array of 16S rDNA sequences in plasma DNA from both CFS and non-fatigued subjects. There were no unique, previously uncharacterized or predominant 16S rDNA sequences in either CFS or non-fatigued subjects. PMID:12498618

  12. Broad energy range neutron spectroscopy using a liquid scintillator and a proportional counter: Application to a neutron spectrum similar to that from an improvised nuclear device

    NASA Astrophysics Data System (ADS)

    Xu, Yanping; Randers-Pehrson, Gerhard; Marino, Stephen A.; Garty, Guy; Harken, Andrew; Brenner, David J.

    2015-09-01

    A novel neutron irradiation facility at the Radiological Research Accelerator Facility (RARAF) has been developed to mimic the neutron radiation from an Improvised Nuclear Device (IND) at relevant distances (e.g. 1.5 km) from the epicenter. The neutron spectrum of this IND-like neutron irradiator was designed according to estimations of the Hiroshima neutron spectrum at 1.5 km. It is significantly different from a standard reactor fission spectrum, because the spectrum changes as the neutrons are transported through air, and it is dominated by neutron energies from 100 keV up to 9 MeV. To verify such wide energy range neutron spectrum, detailed here is the development of a combined spectroscopy system. Both a liquid scintillator detector and a gas proportional counter were used for the recoil spectra measurements, with the individual response functions estimated from a series of Monte Carlo simulations. These normalized individual response functions were formed into a single response matrix for the unfolding process. Several accelerator-based quasi-monoenergetic neutron source spectra were measured and unfolded to test this spectroscopy system. These reference neutrons were produced from two reactions: T(p,n)3He and D(d,n)3He, generating neutron energies in the range between 0.2 and 8 MeV. The unfolded quasi-monoenergetic neutron spectra indicated that the detection system can provide good neutron spectroscopy results in this energy range. A broad-energy neutron spectrum from the 9Be(d,n) reaction using a 5 MeV deuteron beam, measured at 60 degrees to the incident beam was measured and unfolded with the evaluated response matrix. The unfolded broad neutron spectrum is comparable with published time-of-flight results. Finally, the pair of detectors were used to measure the neutron spectrum generated at the RARAF IND-like neutron facility and a comparison is made to the neutron spectrum of Hiroshima.

  13. Broad Energy Range Neutron Spectroscopy using a Liquid Scintillator and a Proportional Counter: Application to a Neutron Spectrum Similar to that from an Improvised Nuclear Device

    PubMed Central

    Randers-Pehrson, Gerhard; Marino, Stephen A.; Garty, Guy; Harken, Andrew; Brenner, David J.

    2015-01-01

    A novel neutron irradiation facility at the Radiological Research Accelerator Facility (RARAF) has been developed to mimic the neutron radiation from an Improvised Nuclear Device (IND) at relevant distances (e.g. 1.5 km) from the epicenter. The neutron spectrum of this IND-like neutron irradiator was designed according to estimations of the Hiroshima neutron spectrum at 1.5 km. It is significantly different from a standard reactor fission spectrum, because the spectrum changes as the neutrons are transported through air, and it is dominated by neutron energies from 100 keV up to 9 MeV. To verify such wide energy range neutron spectrum, detailed here is the development of a combined spectroscopy system. Both a liquid scintillator detector and a gas proportional counter were used for the recoil spectra measurements, with the individual response functions estimated from a series of Monte Carlo simulations. These normalized individual response functions were formed into a single response matrix for the unfolding process. Several accelerator-based quasi-monoenergetic neutron source spectra were measured and unfolded to test this spectroscopy system. These reference neutrons were produced from two reactions: T(p,n)3He and D(d,n)3He, generating neutron energies in the range between 0.2 and 8 MeV. The unfolded quasi-monoenergetic neutron spectra indicated that the detection system can provide good neutron spectroscopy results in this energy range. A broad-energy neutron spectrum from the 9Be(d,n) reaction using a 5 MeV deuteron beam, measured at 60 degrees to the incident beam was measured and unfolded with the evaluated response matrix. The unfolded broad neutron spectrum is comparable with published time-of-flight results. Finally, the pair of detectors were used to measure the neutron spectrum generated at the RARAF IND-like neutron facility and a comparison is made to the neutron spectrum of Hiroshima. PMID:26273118

  14. Broad Energy Range Neutron Spectroscopy using a Liquid Scintillator and a Proportional Counter: Application to a Neutron Spectrum Similar to that from an Improvised Nuclear Device.

    PubMed

    Xu, Yanping; Randers-Pehrson, Gerhard; Marino, Stephen A; Garty, Guy; Harken, Andrew; Brenner, David J

    2015-09-11

    A novel neutron irradiation facility at the Radiological Research Accelerator Facility (RARAF) has been developed to mimic the neutron radiation from an Improvised Nuclear Device (IND) at relevant distances (e.g. 1.5 km) from the epicenter. The neutron spectrum of this IND-like neutron irradiator was designed according to estimations of the Hiroshima neutron spectrum at 1.5 km. It is significantly different from a standard reactor fission spectrum, because the spectrum changes as the neutrons are transported through air, and it is dominated by neutron energies from 100 keV up to 9 MeV. To verify such wide energy range neutron spectrum, detailed here is the development of a combined spectroscopy system. Both a liquid scintillator detector and a gas proportional counter were used for the recoil spectra measurements, with the individual response functions estimated from a series of Monte Carlo simulations. These normalized individual response functions were formed into a single response matrix for the unfolding process. Several accelerator-based quasi-monoenergetic neutron source spectra were measured and unfolded to test this spectroscopy system. These reference neutrons were produced from two reactions: T(p,n)(3)He and D(d,n)(3)He, generating neutron energies in the range between 0.2 and 8 MeV. The unfolded quasi-monoenergetic neutron spectra indicated that the detection system can provide good neutron spectroscopy results in this energy range. A broad-energy neutron spectrum from the (9)Be(d,n) reaction using a 5 MeV deuteron beam, measured at 60 degrees to the incident beam was measured and unfolded with the evaluated response matrix. The unfolded broad neutron spectrum is comparable with published time-of-flight results. Finally, the pair of detectors were used to measure the neutron spectrum generated at the RARAF IND-like neutron facility and a comparison is made to the neutron spectrum of Hiroshima.

  15. Broad-Host-Range ProUSER Vectors Enable Fast Characterization of Inducible Promoters and Optimization of p-Coumaric Acid Production in Pseudomonas putida KT2440.

    PubMed

    Calero, Patricia; Jensen, Sheila I; Nielsen, Alex T

    2016-07-15

    Pseudomonas putida KT2440 has gained increasing interest as a host for the production of biochemicals. Because of the lack of a systematic characterization of inducible promoters in this strain, we generated ProUSER broad-host-expression plasmids that facilitate fast uracil-based cloning. A set of ProUSER-reporter vectors was further created to characterize different inducible promoters. The PrhaB and Pm promoters were orthogonal and showed titratable, high, and homogeneous expression. To optimize the production of p-coumaric acid, P. putida was engineered to prevent degradation of tyrosine and p-coumaric acid. Pm and PrhaB were used to control the expression of a tyrosine ammonia lyase or AroG* and TyrA* involved in tyrosine production, respectively. Pathway expression was optimized by modulating inductions, resulting in small-scale p-coumaric acid production of 1.2 mM, the highest achieved in Pseudomonads under comparable conditions. With broad-host-range compatibility, the ProUSER vectors will serve as useful tools for optimizing gene expression in a variety of bacteria.

  16. Complete Genome sequence of Burkholderia phymatum STM815(T), a broad host range and efficient nitrogen-fixing symbiont of Mimosa species.

    PubMed

    Moulin, Lionel; Klonowska, Agnieszka; Caroline, Bournaud; Booth, Kristina; Vriezen, Jan A C; Melkonian, Rémy; James, Euan K; Young, J Peter W; Bena, Gilles; Hauser, Loren; Land, Miriam; Kyrpides, Nikos; Bruce, David; Chain, Patrick; Copeland, Alex; Pitluck, Sam; Woyke, Tanja; Lizotte-Waniewski, Michelle; Bristow, Jim; Riley, Margaret

    2014-06-15

    Burkholderia phymatum is a soil bacterium able to develop a nitrogen-fixing symbiosis with species of the legume genus Mimosa, and is frequently found associated specifically with Mimosa pudica. The type strain of the species, STM 815(T), was isolated from a root nodule in French Guiana in 2000. The strain is an aerobic, motile, non-spore forming, Gram-negative rod, and is a highly competitive strain for nodulation compared to other Mimosa symbionts, as it also nodulates a broad range of other legume genera and species. The 8,676,562 bp genome is composed of two chromosomes (3,479,187 and 2,697,374 bp), a megaplasmid (1,904,893 bp) and a plasmid hosting the symbiotic functions (595,108 bp).

  17. Complete Genome sequence of Burkholderia phymatum STM815T, a broad host range and efficient nitrogen-fixing symbiont of Mimosa species

    PubMed Central

    Moulin, Lionel; Klonowska, Agnieszka; Caroline, Bournaud; Booth, Kristina; Vriezen, Jan A.C.; Melkonian, Rémy; James, Euan K.; Young, J. Peter W.; Bena, Gilles; Hauser, Loren; Land, Miriam; Kyrpides, Nikos; Bruce, David; Chain, Patrick; Copeland, Alex; Pitluck, Sam; Woyke, Tanja; Lizotte-Waniewski, Michelle; Bristow, Jim; Riley, Margaret

    2014-01-01

    Burkholderia phymatum is a soil bacterium able to develop a nitrogen-fixing symbiosis with species of the legume genus Mimosa, and is frequently found associated specifically with Mimosa pudica. The type strain of the species, STM 815T, was isolated from a root nodule in French Guiana in 2000. The strain is an aerobic, motile, non-spore forming, Gram-negative rod, and is a highly competitive strain for nodulation compared to other Mimosa symbionts, as it also nodulates a broad range of other legume genera and species. The 8,676,562 bp genome is composed of two chromosomes (3,479,187 and 2,697,374 bp), a megaplasmid (1,904,893 bp) and a plasmid hosting the symbiotic functions (595,108 bp). PMID:25197461

  18. Complete Genome sequence of Burkholderia phymatum STM815, a broad host range and efficient nitrogen-fixing symbiont of Mimosa species

    SciTech Connect

    Moulin, Lionel; Klonowska, Agnieszka; Caroline, Bournaud; Booth, Kristina; Vriezen, Jan A.C.; Melkonian, Remy; James, Euan; Young, Peter W.; Bena, Gilles; Hauser, Loren John; Land, Miriam L; Kyrpides, Nikos C; Bruce, David; Chain, Patrick S. G.; Copeland, A; Pitluck, Sam; Woyke, Tanja; Lizotte-Waniewski, Michelle; Bristow, James; Riley, Monica

    2014-01-01

    Burkholderia phymatum is a soil bacterium able to develop a nitrogen-fixing symbiosis with species of the legume genus Mimosa, and is frequently found associated specifically with Mimosa pudica. The type strain of the species, STM 815T, was isolated from a root nodule in French Guiana in 2000. The strain is an aerobic, motile, non-spore forming, Gram-negative rod, and is a highly competitive strain for nodulation compared to other Mimosa symbionts, as it also nodulates a broad range of other legume genera and species. The 8,676,562 bp genome is composed of two chromosomes (3,479,187 and 2,697,374 bp), a megaplasmid (1,904,893 bp) and a plasmid hosting the symbiotic functions (595,108 bp).

  19. Broad-Range Survey of Tick-Borne Pathogens in Southern Germany Reveals a High Prevalence of Babesia microti and a Diversity of Other Tick-Borne Pathogens

    PubMed Central

    Crowder, Chris D.; Carolan, Heather E.; Rounds, Megan A.; Ecker, David J.; Haag, Heike; Mothes, Benedikt; Nolte, Oliver

    2014-01-01

    Abstract Ticks harbor numerous pathogens of significance to human and animal health. A better understanding of the pathogens carried by ticks in a given geographic area can alert health care providers of specific health risks leading to better diagnosis and treatments. In this study, we tested 226 Ixodes ricinis ticks from Southern Germany using a broad-range PCR and electrospray ionization mass spectrometry assay (PCR/ESI-MS) designed to identify tick-borne bacterial and protozoan pathogens in a single test. We found 21.2% of the ticks tested carried Borrelia burgdorferi sensu lato consisting of diverse genospecies; a surprisingly high percentage of ticks were infected with Babesia microti (3.5%). Other organisms found included Borrelia miyamotoi, Rickettsia helvetica, Rickettsia monacensis, and Anaplasma phagocytophilum. Of further significance was our finding that more than 7% of ticks were infected with more than one pathogen or putative pathogen. PMID:25072989

  20. Dry desulfurization of simulated flue gas in a fluidized-bed reactor for a broad range of SO{sub 2} concentration and temperature: A comparison of models

    SciTech Connect

    Suyadal, Y.; Oguz, H.

    1999-08-01

    In this work, dry desulfurization of simulated flue gas was investigated in a batchwise operated laboratory-scale stainless steel fluidized-bed reactor (46 x 500 mm{sup 2}) by using calcium-containing local Turkish limestone (Karaagach/MUS) which was calcined at 900 C with 5% H{sub 2}O vapor. The sulfation reaction was carried out in a broad range of temperature (200 {le} T({degree}C) {le} 900) and SO{sub 2} feedstock concentration (1000 {le} C(ppm SO{sub 2}) {le} 6000). The experimental sulfation conversion-time data were tested according to unreacted shrinking core model (SCM), changing the grain size model (GM) and random pore model (RPM). It was found that the random pore model with control of product layer (CaSO{sub 3}/CaSO{sub 4}) diffusion described the experimental data best.

  1. Characterization of 6H-SiC JFET Integrated Circuits Over A Broad Temperature Range from -150 C to +500 C

    NASA Technical Reports Server (NTRS)

    Neudeck, Philip G.; Krasowski, Michael J.; Chen, Liang-Yu; Prokop, Norman F.

    2009-01-01

    The NASA Glenn Research Center has previously reported prolonged stable operation of simple prototype 6H-SiC JFET integrated circuits (logic gates and amplifier stages) for thousands of hours at +500 C. This paper experimentally investigates the ability of these 6H-SiC JFET devices and integrated circuits to also function at cold temperatures expected to arise in some envisioned applications. Prototype logic gate ICs experimentally demonstrated good functionality down to -125 C without changing circuit input voltages. Cascaded operation of gates at cold temperatures was verified by externally wiring gates together to form a 3-stage ring oscillator. While logic gate output voltages exhibited little change across the broad temperature range from -125 C to +500 C, the change in operating frequency and power consumption of these non-optimized logic gates as a function of temperature was much larger and tracked JFET channel conduction properties.

  2. Characterization of 6H-SiC JFET Integrated Circuits Over A Broad Temperature Range from -150 C to +500 C

    NASA Technical Reports Server (NTRS)

    Neudeck, Philip G.; Krasowski, Michael J.; Chen, Liang-Yu; Prokop, Norman F.

    2009-01-01

    The NASA Glenn Research Center has previously reported prolonged stable operation of simple prototype 6H-SiC JFET integrated circuits (logic gates and amplifier stages) for thousands of hours at +500 C. This paper experimentally investigates the ability of these 6H-SiC JFET devices and integrated circuits to also function at cold temperatures expected to arise in some envisioned applications. Prototype logic gate ICs experimentally demonstrated good functionality down to -125 C without changing circuit input voltages. Cascaded operation of gates at cold temperatures was verified by externally wiring gates together to form a 3-stage ring oscillator. While logic gate output voltages exhibited little change across the broad temperature range from -125 C to +500 C, the change in operating frequency and power consumption of these non-optimized logic gates as a function of temperature was much larger and tracked JFET channel conduction properties.

  3. The Detection and Sequencing of a Broad-Host-Range Conjugative IncP-1β Plasmid in an Epidemic Strain of Mycobacterium abscessus subsp. bolletii

    PubMed Central

    Leão, Sylvia Cardoso; Matsumoto, Cristianne Kayoko; Carneiro, Adriana; Ramos, Rommel Thiago; Nogueira, Christiane Lourenço; Junior, James Daltro Lima; Lima, Karla Valéria; Lopes, Maria Luiza; Schneider, Horacio; Azevedo, Vasco Ariston; da Costa da Silva, Artur

    2013-01-01

    Background An extended outbreak of mycobacterial surgical infections occurred in Brazil during 2004–2008. Most infections were caused by a single strain of Mycobacterium abscessus subsp. bolletii, which was characterized by a specific rpoB sequevar and two highly similar pulsed-field gel electrophoresis (PFGE) patterns differentiated by the presence of a ∼50 kb band. The nature of this band was investigated. Methodology/Principal Findings Genomic sequencing of the prototype outbreak isolate INCQS 00594 using the SOLiD platform demonstrated the presence of a 56,264-bp circular plasmid, designated pMAB01. Identity matrices, genetic distances and phylogeny analyses indicated that pMAB01 belongs to the broad-host-range plasmid subgroup IncP-1β and is highly related to BRA100, pJP4, pAKD33 and pB10. The presence of pMAB01-derived sequences in 41 M. abscessus subsp. bolletii isolates was evaluated using PCR, PFGE and Southern blot hybridization. Sixteen of the 41 isolates showed the presence of the plasmid. The plasmid was visualized as a ∼50-kb band using PFGE and Southern blot hybridization in 12 isolates. The remaining 25 isolates did not exhibit any evidence of this plasmid. The plasmid was successfully transferred to Escherichia coli by conjugation and transformation. Lateral transfer of pMAB01 to the high efficient plasmid transformation strain Mycobacterium smegmatis mc2155 could not be demonstrated. Conclusions/Significance The occurrence of a broad-host-range IncP-1β plasmid in mycobacteria is reported for the first time. Thus, genetic exchange could result in the emergence of specific strains that might be better adapted to cause human disease. PMID:23565273

  4. Miropin, a novel bacterial serpin from the periodontopathogen Tannerella forsythia, inhibits a broad range of proteases by using different peptide bonds within the reactive center loop.

    PubMed

    Ksiazek, Miroslaw; Mizgalska, Danuta; Enghild, Jan J; Scavenius, Carsten; Thogersen, Ida B; Potempa, Jan

    2015-01-02

    All prokaryotic genes encoding putative serpins identified to date are found in environmental and commensal microorganisms, and only very few prokaryotic serpins have been investigated from a mechanistic standpoint. Herein, we characterized a novel serpin (miropin) from the human pathogen Tannerella forsythia, a bacterium implicated in initiation and progression of human periodontitis. In contrast to other serpins, miropin efficiently inhibited a broad range of proteases (neutrophil and pancreatic elastases, cathepsin G, subtilisin, and trypsin) with a stoichiometry of inhibition of around 3 and second-order association rate constants that ranged from 2.7 × 10(4) (cathepsin G) to 7.1 × 10(5) m(-1)s(-1) (subtilisin). Inhibition was associated with the formation of complexes that were stable during SDS-PAGE. The unusually broad specificity of miropin for target proteases is achieved through different active sites within the reactive center loop upstream of the P1-P1' site, which was predicted from an alignment of the primary structure of miropin with those of well studied human and prokaryotic serpins. Thus, miropin is unique among inhibitory serpins, and it has apparently evolved the ability to inhibit a multitude of proteases at the expense of a high stoichiometry of inhibition and a low association rate constant. These characteristics suggest that miropin arose as an adaptation to the highly proteolytic environment of subgingival plaque, which is exposed continually to an array of host proteases in the inflammatory exudate. In such an environment, miropin may function as an important virulence factor by protecting bacterium from the destructive activity of neutrophil serine proteases. Alternatively, it may act as a housekeeping protein that regulates the activity of endogenous T. forsythia serine proteases.

  5. A Thermostable Glucoamylase from Bispora sp. MEY-1 with Stability over a Broad pH Range and Significant Starch Hydrolysis Capacity

    PubMed Central

    Bai, Yingguo; Wang, Kun; Niu, Canfang; Huang, Huoqing; Shi, Pengjun; Wang, Caihong; Yang, Peilong; Yao, Bin

    2014-01-01

    Background Glucoamylase is an exo-type enzyme that converts starch completely into glucose from the non-reducing ends. To meet the industrial requirements for starch processing, a glucoamylase with excellent thermostability, raw-starch degradation ability and high glucose yield is much needed. In the present study we selected the excellent Carbohydrate-Activity Enzyme (CAZyme) producer, Bispora sp. MEY-1, as the microbial source for glucoamylase gene exploitation. Methodology/Principal Findings A glucoamylase gene (gla15) was cloned from Bispora sp. MEY-1 and successfully expressed in Pichia pastoris with a high yield of 34.1 U/ml. Deduced GLA15 exhibits the highest identity of 64.2% to the glucoamylase from Talaromyces (Rasamsonia) emersonii. Purified recombinant GLA15 was thermophilic and showed the maximum activity at 70°C. The enzyme was stable over a broad pH range (2.2–11.0) and at high temperature up to 70°C. It hydrolyzed the breakages of both α-1,4- and α-1,6-glycosidic linkages in amylopectin, soluble starch, amylose, and maltooligosaccharides, and had capacity to degrade raw starch. TLC and H1-NMR analysis showed that GLA15 is a typical glucoamylase of GH family 15 that releases glucose units from the non-reducing ends of α-glucans. The combination of Bacillus licheniformis amylase and GLA15 hydrolyzed 96.14% of gelatinized maize starch after 6 h incubation, which was about 9% higher than that of the combination with a commercial glucoamylase from Aspergillus niger. Conclusion/Significance GLA15 has a broad pH stability range, high-temperature thermostability, high starch hydrolysis capacity and high expression yield. In comparison with the commercial glucoamylase from A. niger, GLA15 represents a better candidate for application in the food industry including production of glucose, glucose syrups, and high-fructose corn syrups. PMID:25415468

  6. Novel Virulent and Broad-Host-Range Erwinia amylovora Bacteriophages Reveal a High Degree of Mosaicism and a Relationship to Enterobacteriaceae Phages ▿†

    PubMed Central

    Born, Yannick; Fieseler, Lars; Marazzi, Janine; Lurz, Rudi; Duffy, Brion; Loessner, Martin J.

    2011-01-01

    A diverse set of 24 novel phages infecting the fire blight pathogen Erwinia amylovora was isolated from fruit production environments in Switzerland. Based on initial screening, four phages (L1, M7, S6, and Y2) with broad host ranges were selected for detailed characterization and genome sequencing. Phage L1 is a member of the Podoviridae, with a 39.3-kbp genome featuring invariable genome ends with direct terminal repeats. Phage S6, another podovirus, was also found to possess direct terminal repeats but has a larger genome (74.7 kbp), and the virus particle exhibits a complex tail fiber structure. Phages M7 and Y2 both belong to the Myoviridae family and feature long, contractile tails and genomes of 84.7 kbp (M7) and 56.6 kbp (Y2), respectively, with direct terminal repeats. The architecture of all four phage genomes is typical for tailed phages, i.e., organized into function-specific gene clusters. All four phages completely lack genes or functions associated with lysogeny control, which correlates well with their broad host ranges and indicates strictly lytic (virulent) lifestyles without the possibility for host lysogenization. Comparative genomics revealed that M7 is similar to E. amylovora virus ΦEa21-4, whereas L1, S6, and Y2 are unrelated to any other E. amylovora phage. Instead, they feature similarities to enterobacterial viruses T7, N4, and ΦEcoM-GJ1. In a series of laboratory experiments, we provide proof of concept that specific two-phage cocktails offer the potential for biocontrol of the pathogen. PMID:21764969

  7. Transcript level of erythroid differentiation-related factor, a candidate surrogate marker for transmissible spongiform encephalopathy diseases in blood, shows a broad range of variation in healthy individuals.

    PubMed

    Glock, Barbara; Winter, Maya; Rennhofer, Simone O; Brunhölzl, Elisabeth; Tröscher, Doris; Reisacher, Rosemarie B K; Mayr, Wolfgang R

    2003-12-01

    In 2001, it was demonstrated that the expression of the erythroid differentiation-related factor (EDRF) is reduced in lymphatic tissues of rodents and cattle as well as in whole blood of sheep that suffer from transmissible spongiform encephalopathy. To determine whether the normal range of EDRF expression varies in healthy individuals, mRNA levels were measured in whole blood samples from 106 healthy blood donors by quantitative real-time RT-PCR. Furthermore, the correlations of transcript levels with individual physical characteristics were analyzed. In addition, EDRF expression was examined in total RNA samples from a lymph node and the intestine. The data show that EDRF mRNA levels in healthy persons vary within a total range of 2 log units as well as they display a weak correlation with body height. Furthermore, it was found that EDRF is also expressed in lymph nodes and the intestine. Owing to its broad range of variation, measuring the EDRF expression does not seem to be a good surrogate marker, unless an altered expression is distinctively different from the varying level in healthy humans.

  8. Diagnosis of Bacterial Bloodstream Infections: A 16S Metagenomics Approach.

    PubMed

    Decuypere, Saskia; Meehan, Conor J; Van Puyvelde, Sandra; De Block, Tessa; Maltha, Jessica; Palpouguini, Lompo; Tahita, Marc; Tinto, Halidou; Jacobs, Jan; Deborggraeve, Stijn

    2016-02-01

    Bacterial bloodstream infection (bBSI) is one of the leading causes of death in critically ill patients and accurate diagnosis is therefore crucial. We here report a 16S metagenomics approach for diagnosing and understanding bBSI. The proof-of-concept was delivered in 75 children (median age 15 months) with severe febrile illness in Burkina Faso. Standard blood culture and malaria testing were conducted at the time of hospital admission. 16S metagenomics testing was done retrospectively and in duplicate on the blood of all patients. Total DNA was extracted from the blood and the V3-V4 regions of the bacterial 16S rRNA genes were amplified by PCR and deep sequenced on an Illumina MiSeq sequencer. Paired reads were curated, taxonomically labeled, and filtered. Blood culture diagnosed bBSI in 12 patients, but this number increased to 22 patients when combining blood culture and 16S metagenomics results. In addition to superior sensitivity compared to standard blood culture, 16S metagenomics revealed important novel insights into the nature of bBSI. Patients with acute malaria or recovering from malaria had a 7-fold higher risk of presenting polymicrobial bloodstream infections compared to patients with no recent malaria diagnosis (p-value = 0.046). Malaria is known to affect epithelial gut function and may thus facilitate bacterial translocation from the intestinal lumen to the blood. Importantly, patients with such polymicrobial blood infections showed a 9-fold higher risk factor for not surviving their febrile illness (p-value = 0.030). Our data demonstrate that 16S metagenomics is a powerful approach for the diagnosis and understanding of bBSI. This proof-of-concept study also showed that appropriate control samples are crucial to detect background signals due to environmental contamination.

  9. Diagnosis of Bacterial Bloodstream Infections: A 16S Metagenomics Approach

    PubMed Central

    Van Puyvelde, Sandra; De Block, Tessa; Maltha, Jessica; Palpouguini, Lompo; Tahita, Marc; Tinto, Halidou; Jacobs, Jan; Deborggraeve, Stijn

    2016-01-01

    Background Bacterial bloodstream infection (bBSI) is one of the leading causes of death in critically ill patients and accurate diagnosis is therefore crucial. We here report a 16S metagenomics approach for diagnosing and understanding bBSI. Methodology/Principal Findings The proof-of-concept was delivered in 75 children (median age 15 months) with severe febrile illness in Burkina Faso. Standard blood culture and malaria testing were conducted at the time of hospital admission. 16S metagenomics testing was done retrospectively and in duplicate on the blood of all patients. Total DNA was extracted from the blood and the V3–V4 regions of the bacterial 16S rRNA genes were amplified by PCR and deep sequenced on an Illumina MiSeq sequencer. Paired reads were curated, taxonomically labeled, and filtered. Blood culture diagnosed bBSI in 12 patients, but this number increased to 22 patients when combining blood culture and 16S metagenomics results. In addition to superior sensitivity compared to standard blood culture, 16S metagenomics revealed important novel insights into the nature of bBSI. Patients with acute malaria or recovering from malaria had a 7-fold higher risk of presenting polymicrobial bloodstream infections compared to patients with no recent malaria diagnosis (p-value = 0.046). Malaria is known to affect epithelial gut function and may thus facilitate bacterial translocation from the intestinal lumen to the blood. Importantly, patients with such polymicrobial blood infections showed a 9-fold higher risk factor for not surviving their febrile illness (p-value = 0.030). Conclusions/Significance Our data demonstrate that 16S metagenomics is a powerful approach for the diagnosis and understanding of bBSI. This proof-of-concept study also showed that appropriate control samples are crucial to detect background signals due to environmental contamination. PMID:26927306

  10. Bacillus polymachus sp. nov., with a broad range of antibacterial activity, isolated from forest topsoil samples by using a modified culture method.

    PubMed

    Nguyen, Tuan Manh; Kim, Jaisoo

    2015-02-01

    A new, modified culture method that utilizes a transwell plate with a 0.4 µm pore-size microporous membrane was developed. This system allows only trace nutrients from the soil into the liquid culture through the microporous membrane. The method is a more powerful tool for the discovery of novel species from soils than are traditional methods. Such newly identified species could potentially produce useful metabolites. A bacterial strain, T515(T), was isolated using this modified culture method. Growth of strain T515(T) occurred at pH 4-9 in a temperature range between 20 °C and 40 °C and in the presence of 0-2 % (w/v) NaCl on R2A agar. Colonies on the agar plates were tiny, white, and convex after 5 days incubation at 28 °C. Comparative analysis of the nearly full-length 16S rRNA gene sequence of strain T515(T) revealed close pairwise similarity with species of the genus Bacillus, and strain T515(T) was most closely related to Bacillus panaciterrae Gsoil 1517(T) (96.7 %) and Bacillus funiculus NAF001(T) (96.0 %). The major quinone of strain T515(T) was menaquinone-7 (MK-7) and the major fatty acids were iso-C15 : 0 (45.5 %), anteiso-C15 : 0 (23.2 %) and C16 : 0 (10.9 %). The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Strain T515(T) was sensitive to streptomycin and tetracycline, but resistant to rifampicin (0.125 µg ml(-1)), ampicillin (0.5 µg ml(-1)) and chloramphenicol (1 µg ml(-1)). The strain showed antimicrobial activities against the six strains tested: Bacillus subtilis KEMB 51201-001, Staphylococcus aureus KEMB 4659, Pseudomonas aeruginosa KACC 10185, Staphylococcus epidermidis KACC 13234, Paenibacillus larvae KACC 14031 and Escherichia coli KEMB 212-234. Based on these results, strain T515(T) represents a novel species of the genus Bacillus with the proposed name, Bacillus polymachus sp. nov. The type strain is T515(T) ( = KEMB 9005-168(T) = KACC 18242(T) = NBRC 110614(T)).

  11. Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein.

    PubMed

    Dammeyer, Thorben; Timmis, Kenneth N; Tinnefeld, Philip

    2013-05-20

    In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains. Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs. The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co-localization studies. In addition, the

  12. Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein

    PubMed Central

    2013-01-01

    Background In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains. Results Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs. Conclusion The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co

  13. Monoclonal antibodies isolated from human B cells neutralize a broad range of H1 subtype influenza A viruses including swine-origin Influenza virus (S-OIV).

    PubMed

    Burioni, Roberto; Canducci, Filippo; Mancini, Nicasio; Clementi, Nicola; Sassi, Monica; De Marco, Donata; Diotti, Roberta Antonia; Saita, Diego; Sampaolo, Michela; Sautto, Giuseppe; Pianezze, Matteo; Clementi, Massimo

    2010-03-30

    The new H1N1 swine-origin influenza virus (S-OIV) strain is a global health problem. The elucidation of the virus-host relationship is crucial for the control of the new infection. Two human monoclonal antibody Fab fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned before the emergence of S-OIV from a patient who had a broad-range H1N1 serum neutralizing activity. The two HMabs neutralized all tested H1N1 strains, including S-OIV and a swine strain with IC(50) ranging from 2 to 7 microg/ml. Data demonstrate that infection with previously circulating H1N1 strains can elicit antibodies neutralizing S-OIV. Finally, the human genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered being potentially useful in the prophylaxis and the treatment of this human infection.

  14. Crystal structure of a trimeric archaeal adenylate kinase from the mesophile Methanococcus maripaludis with an unusually broad functional range and thermal stability.

    PubMed

    Davlieva, Milya; Shamoo, Yousif

    2010-02-01

    The structure of the trimeric adenylate kinase from the Archaebacteria Methanococcus mariplaludis (AK(MAR)) has been solved to 2.5-A resolution and the temperature dependent stability and kinetics of the enzyme measured. The K(M) and V(max) of AK(MAR) exhibit only modest temperature dependence from 30 degrees -60 degrees C. Although M. mariplaludis is a mesophile with a maximum growth temperature of 43 degrees C, AK(MAR) has a very broad functional range and stability (T(m) = 74.0 degrees C) that are more consistent with a thermophilic enzyme with high thermostability and exceptional activity over a wide range of temperatures, suggesting that this microbe may have only recently invaded a mesophilic niche and has yet to fully adapt. A comparison of the Local Structural Entropy (LSE) for AK(MAR) to the related adenylate kinases from the mesophile Methanococcus voltae and thermophile Methanococcus thermolithotrophicus show that changes in LSE are able to fully account for the intermediate stability of AK(MAR) and highlights a general mechanism for protein adaptation in this class of enzymes.

  15. Predicting the Remaining Lifespan and Cultivation-Related Loss of Osteogenic Capacity of Bone Marrow Multipotential Stromal Cells Applicable across a Broad Donor Age Range

    PubMed Central

    Churchman, Sarah M.; Boxall, Sally A.; McGonagle, Dennis

    2017-01-01

    Background and Objectives. Culture expanded multipotential stromal cells (MSCs) have considerable potential for bone regeneration therapy but their wider use is constrained by the lack of simple and predictive assays of functional potency. Extended passaging leads to loss of multipotency but speed of decline depends on MSC donor age. The aim of this study was to develop an assay predictive of MSC culture longevity applicable to a broad donor age range. Materials and Methods. Bone marrow (BM, n = 7) was obtained from a diverse range (2–72 years) of healthy donors. MSCs were culture expanded to senescence and their osteoprogenitor content, gene expression profiles, epigenetic signature, and telomere behaviour were measured throughout. Output data was combined for modelling purposes. Results. Regardless of donor age, cultures' osteoprogenitor content correlated better with remaining lifespan (population doublings before senescence, PD-BS) than proliferative history (accrued PDs). Individual gene's expression or telomere length did not predict PD-BS but methylation of individual CpG islands did, PRAMEF2 in particular (r = 0.775). Coupling the steep relationship of relative SPARC expression with PD-BS (r = −0.753) the formula SPARC × 1/PREMEF2 gave an improved correlation (r = −0.893). Conclusion. A formula based on SPARC mRNA and PRAMEF2 methylation may be used to predict remaining BM-MSC longevity and related loss of multipotentiality independent of donor age. PMID:28298930

  16. VASCULAR-RELATED NAC-DOMAIN7 directly regulates the expression of a broad range of genes for xylem vessel formation.

    PubMed

    Yamaguchi, Masatoshi; Mitsuda, Nobutaka; Ohtani, Misato; Ohme-Takagi, Masaru; Kato, Ko; Demura, Taku

    2011-05-01

    The Arabidopsis thaliana NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), acts as a key regulator of xylem vessel differentiation. In order to identify direct target genes of VND7, we performed global transcriptome analysis using Arabidopsis transgenic lines in which VND7 activity could be induced post-translationally. This analysis identified 63 putative direct target genes of VND7, which encode a broad range of proteins, such as transcription factors, IRREGULAR XYLEM proteins and proteolytic enzymes, known to be closely associated with xylem vessel formation. Recombinant VND7 protein binds to several promoter sequences present in candidate direct target genes: specifically, in the promoter of XYLEM CYSTEINE PEPTIDASE1, two distinct regions were demonstrated to be responsible for VND7 binding. We also found that expression of VND7 restores secondary cell wall formation in the fiber cells of inflorescence stems of nst1 nst3 double mutants, as well as expression of NAC SECONDARY WALL THICKENING PROMOTING FACTOR3 (NST3, however, the vessel-type secondary wall deposition was observed only as a result of VND7 expression. These findings indicated that VND7 upregulates, directly and/or indirectly, many genes involved in a wide range of processes in xylem vessel differentiation, and that its target genes are partially different from those of NSTs. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

  17. Predicting the Remaining Lifespan and Cultivation-Related Loss of Osteogenic Capacity of Bone Marrow Multipotential Stromal Cells Applicable across a Broad Donor Age Range.

    PubMed

    Churchman, Sarah M; Boxall, Sally A; McGonagle, Dennis; Jones, Elena A

    2017-01-01

    Background and Objectives. Culture expanded multipotential stromal cells (MSCs) have considerable potential for bone regeneration therapy but their wider use is constrained by the lack of simple and predictive assays of functional potency. Extended passaging leads to loss of multipotency but speed of decline depends on MSC donor age. The aim of this study was to develop an assay predictive of MSC culture longevity applicable to a broad donor age range. Materials and Methods. Bone marrow (BM, n = 7) was obtained from a diverse range (2-72 years) of healthy donors. MSCs were culture expanded to senescence and their osteoprogenitor content, gene expression profiles, epigenetic signature, and telomere behaviour were measured throughout. Output data was combined for modelling purposes. Results. Regardless of donor age, cultures' osteoprogenitor content correlated better with remaining lifespan (population doublings before senescence, PD-BS) than proliferative history (accrued PDs). Individual gene's expression or telomere length did not predict PD-BS but methylation of individual CpG islands did, PRAMEF2 in particular (r = 0.775). Coupling the steep relationship of relative SPARC expression with PD-BS (r = -0.753) the formula SPARC × 1/PREMEF2 gave an improved correlation (r = -0.893). Conclusion. A formula based on SPARC mRNA and PRAMEF2 methylation may be used to predict remaining BM-MSC longevity and related loss of multipotentiality independent of donor age.

  18. Broad Habitat Range of the Phylogenetically Narrow R-BT065 Cluster, Representing a Core Group of the Betaproteobacterial Genus Limnohabitans▿ †

    PubMed Central

    Šimek, Karel; Kasalický, Vojtěch; Jezbera, Jan; Jezberová, Jitka; Hejzlar, Josef; Hahn, Martin W.

    2010-01-01

    The distribution of the phylogenetically narrow R-BT065 cluster (Betaproteobacteria) in 102 freshwater lakes, reservoirs, and various ponds located in central Europe (a total of 122 samples) was examined by using a cluster-specific fluorescence in situ hybridization probe. These habitats differ markedly in pH, conductivity, trophic status, surface area, altitude, bedrock type, and other limnological characteristics. Despite the broad ecological diversity of the habitats investigated, the cluster was detected in 96.7% of the systems, and its occurrence was not restricted to a certain habitat type. However, the relative proportions of the cluster in the total bacterioplankton were significantly lower in humic and acidified lakes than in pH-neutral or alkaline habitats. On average, the cluster accounted for 9.4% of the total bacterioplankton (range, 0 to 29%). The relative abundance and absolute abundance of these bacteria were significantly and positively related to higher pH, conductivity, and the proportion of low-molecular-weight compounds in dissolved organic carbon (DOC) and negatively related to the total DOC and dissolved aromatic carbon contents. Together, these parameters explained 55.3% of the variability in the occurrence of the cluster. Surprisingly, no clear relationship of the R-BT065 bacteria to factors indicating the trophic status of habitats (i.e., different forms of phosphorus and chlorophyll a content) was found. Based on our results and previously published data, we concluded that the R-BT065 cluster represents a ubiquitous, highly active segment of bacterioplankton in nonacidic lakes and ponds and that alga-derived substrates likely form the main pool of substrates responsible for its high growth potential and broad distribution in freshwater habitats. PMID:19948856

  19. Broad habitat range of the phylogenetically narrow R-BT065 cluster, representing a core group of the Betaproteobacterial genus Limnohabitans.

    PubMed

    Simek, Karel; Kasalicky, Vojtech; Jezbera, Jan; Jezberová, Jitka; Hejzlar, Josef; Hahn, Martin W

    2010-02-01

    The distribution of the phylogenetically narrow R-BT065 cluster (Betaproteobacteria) in 102 freshwater lakes, reservoirs, and various ponds located in central Europe (a total of 122 samples) was examined by using a cluster-specific fluorescence in situ hybridization probe. These habitats differ markedly in pH, conductivity, trophic status, surface area, altitude, bedrock type, and other limnological characteristics. Despite the broad ecological diversity of the habitats investigated, the cluster was detected in 96.7% of the systems, and its occurrence was not restricted to a certain habitat type. However, the relative proportions of the cluster in the total bacterioplankton were significantly lower in humic and acidified lakes than in pH-neutral or alkaline habitats. On average, the cluster accounted for 9.4% of the total bacterioplankton (range, 0 to 29%). The relative abundance and absolute abundance of these bacteria were significantly and positively related to higher pH, conductivity, and the proportion of low-molecular-weight compounds in dissolved organic carbon (DOC) and negatively related to the total DOC and dissolved aromatic carbon contents. Together, these parameters explained 55.3% of the variability in the occurrence of the cluster. Surprisingly, no clear relationship of the R-BT065 bacteria to factors indicating the trophic status of habitats (i.e., different forms of phosphorus and chlorophyll a content) was found. Based on our results and previously published data, we concluded that the R-BT065 cluster represents a ubiquitous, highly active segment of bacterioplankton in nonacidic lakes and ponds and that alga-derived substrates likely form the main pool of substrates responsible for its high growth potential and broad distribution in freshwater habitats.

  20. Reconstructing 16S rRNA genes in metagenomic data.

    PubMed

    Yuan, Cheng; Lei, Jikai; Cole, James; Sun, Yanni

    2015-06-15

    Metagenomic data, which contains sequenced DNA reads of uncultured microbial species from environmental samples, provide a unique opportunity to thoroughly analyze microbial species that have never been identified before. Reconstructing 16S ribosomal RNA, a phylogenetic marker gene, is usually required to analyze the composition of the metagenomic data. However, massive volume of dataset, high sequence similarity between related species, skewed microbial abundance and lack of reference genes make 16S rRNA reconstruction difficult. Generic de novo assembly tools are not optimized for assembling 16S rRNA genes. In this work, we introduce a targeted rRNA assembly tool, REAGO (REconstruct 16S ribosomal RNA Genes from metagenOmic data). It addresses the above challenges by combining secondary structure-aware homology search, zproperties of rRNA genes and de novo assembly. Our experimental results show that our tool can correctly recover more rRNA genes than several popular generic metagenomic assembly tools and specially designed rRNA construction tools. The source code of REAGO is freely available at https://github.com/chengyuan/reago. © The Author 2015. Published by Oxford University Press.

  1. Elevated Norepinephrine may be a Unifying Etiological Factor in the Abuse of a Broad Range of Substances: Alcohol, Nicotine, Marijuana, Heroin, Cocaine, and Caffeine.

    PubMed

    Fitzgerald, Paul J

    2013-10-13

    A wide range of commonly abused drugs have effects on the noradrenergic neurotransmitter system, including alterations during acute intoxication and chronic use of these drugs. It is not established, however, that individual differences in noradrenergic signaling, which may be present prior to use of drugs, predispose certain persons to substance abuse. This paper puts forth the novel hypothesis that elevated noradrenergic signaling, which may be raised largely due to genetics but also due to environmental factors, is an etiological factor in the abuse of a wide range of substances, including alcohol, nicotine, marijuana, heroin, cocaine, and caffeine. Data are reviewed for each of these drugs comprising their interaction with norepinephrine during acute intoxication, long-term use, subsequent withdrawal, and stress-induced relapse. In general, the data suggest that these drugs acutely boost noradrenergic signaling, whereas long-term use also affects this neurotransmitter system, possibly suppressing it. During acute withdrawal after chronic drug use, noradrenergic signaling tends to be elevated, consistent with the observation that norepinephrine lowering drugs such as clonidine reduce withdrawal symptoms. Since psychological stress can promote relapse of drug seeking in susceptible individuals and stress produces elevated norepinephrine release, this suggests that these drugs may be suppressing noradrenergic signaling during chronic use or instead elevating it only in reward circuits of the brain. If elevated noradrenergic signaling is an etiological factor in the abuse of a broad range of substances, then chronic use of pharmacological agents that reduce noradrenergic signaling, such as clonidine, guanfacine, lofexidine, propranolol, or prazosin, may help prevent or treat drug abuse in general.

  2. A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase.

    PubMed

    Podetz-Pedersen, Kelly M; Olson, Erik R; Somia, Nikunj V; Russell, Stephen J; McIvor, R Scott

    2016-01-19

    The Sleeping Beauty (SB) transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x) at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg), we observed a broad range of pCMV-SB100x doses (0.1-2.5 µg) conferring optimal levels of long-term expression (>10(11) photons/second/cm(2)). At a fixed dose of 0.5 μg of pCMV-SB100x, maximal long-term luciferase expression (>10(10) photons/second/cm(2)) was achieved at a transposon dose of 5-125 μg. We also found that in the linear range of transposon doses (100 ng), co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver.

  3. A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase

    PubMed Central

    Podetz-Pedersen, Kelly M; Olson, Erik R; Somia, Nikunj V; Russell, Stephen J; McIvor, R Scott

    2016-01-01

    The Sleeping Beauty (SB) transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x) at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg), we observed a broad range of pCMV-SB100x doses (0.1–2.5 µg) conferring optimal levels of long-term expression (>1011 photons/second/cm2). At a fixed dose of 0.5 μg of pCMV-SB100x, maximal long-term luciferase expression (>1010 photons/second/cm2) was achieved at a transposon dose of 5–125 μg. We also found that in the linear range of transposon doses (100 ng), co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver. PMID:26784638

  4. Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria.

    PubMed

    Zhang, Jia Jia; Tang, Xiaoyu; Zhang, Michelle; Nguyen, Darlene; Moore, Bradley S

    2017-09-05

    Heterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-host-range expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC from Pseudoalteromonas luteoviolacea 2ta16 revealed robust production in two proteobacterial hosts, Pseudomonas putida KT2440 and Agrobacterium tumefaciens LBA4404, but very little production of the antibiotic in various laboratory strains of Escherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor from P. luteoviolacea 2ta16, PviR, that increases pathway transcription and violacein production in E. coli by ∼60-fold independently of acyl-homoserine lactone autoinducers. Although E. coli harbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of various E. coli transcription factors did not result in a statistically significant increase in violacein production, while overexpression of two A. tumefaciens PviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility.IMPORTANCE Although Gram-positive heterologous hosts such as Streptomyces have been developed and optimized to support diverse secondary metabolic reactions, there has been comparatively less work on Gram-negative hosts, some of which grow faster and are easier to work with. This work presents a new genetic platform for direct cloning and broad-host-range heterologous expression of BGCs

  5. ContEst16S: an algorithm that identifies contaminated prokaryotic genomes using 16S RNA gene sequences.

    PubMed

    Lee, Imchang; Chalita, Mauricio; Ha, Sung-Min; Na, Seong-In; Yoon, Seok-Hwan; Chun, Jongsik

    2017-06-01

    Thanks to the recent advancement of DNA sequencing technology, the cost and time of prokaryotic genome sequencing have been dramatically decreased. It has repeatedly been reported that genome sequencing using high-throughput next-generation sequencing is prone to contaminations due to its high depth of sequencing coverage. Although a few bioinformatics tools are available to detect potential contaminations, these have inherited limitations as they only use protein-coding genes. Here we introduce a new algorithm, called ContEst16S, to detect potential contaminations using 16S rRNA genes from genome assemblies. We screened 69 745 prokaryotic genomes from the NCBI Assembly Database using ContEst16S and found that 594 were contaminated by bacteria, human and plants. Of the predicted contaminated genomes, 8 % were not predicted by the existing protein-coding gene-based tool, implying that both methods can be complementary in the detection of contaminations. A web-based service of the algorithm is available at www.ezbiocloud.net/tools/contest16s.

  6. Contraception for the under 16s: better safe than sorry.

    PubMed

    Cook, A

    1981-09-16

    acceptible if the couple was engaged, and 5.4% were totally against it, 9) 62% felt abortion was the right of every woman and 31.1% felt it was acceptible if the physical or mental well being of the mother was at risk, 10) 40.9% agreed with the British Medical Association policy on teenage contraception which advises doctors to encourage under 16's to tell their parents, but if they refuse, the doctor can still prescribe the pill, 11) 22.7% wanted contraception unconditionally available, 18.2% felt it should be dependent on parental knowledge, and 17% said it should not be available, 12) there was a trend for opinions to become less liberal as age increased, and 13) young girls appear to be less conscientious in using contraception than older women.

  7. Evaluation of viral extraction methods on a broad range of Ready-To-Eat foods with conventional and real-time RT-PCR for Norovirus GII detection.

    PubMed

    Baert, Leen; Uyttendaele, Mieke; Debevere, Johan

    2008-03-31

    Noroviruses (NoV) are a common cause of foodborne outbreaks. In spite of that, no standard viral detection method is available for food products. Therefore, three viral elution-concentration methods and one direct RNA isolation method were evaluated on a broad range of Ready-To-Eat (RTE) food products (mixed lettuce, fruit salad, raspberries and two RTE dishes) artificially seeded with a diluted stool sample contaminated with NoV genogroup II. These seeding experiments revealed two categories of RTE products, fruits and vegetables grouped together and RTE dishes (penne and tagliatelle salads) which are rich in proteins and fat formed another category. The RNA extracts were amplified and detected with two conventional RT-PCR systems (Booster and Semi-nested GII) and one real-time RT-PCR (Real-time GII) assay. A fast direct RNA isolation method detected 10(2) RT-PCRU on 10 g penne and tagliatelle salads with the conventional RT-PCR assays. However real-time RT-PCR was less sensitive for penne salad. A viral elution-concentration method, including a buffer solution for the elution step and one polyethylene glycol (PEG) precipitation step, was able to detect 10(2) RT-PCRU on 50 g frozen raspberries with conventional and real-time RT-PCR assays. Moreover the latter extraction method used no environmental hazardous chemical reagents and was easy to perform.

  8. Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors.

    PubMed Central

    Simon, R; Hötte, B; Klauke, B; Kosier, B

    1991-01-01

    On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed. Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris. More than 20 different IS elements were isolated and characterized. The 16 IS elements from Rhizobium meliloti were further used to characterize various R. meliloti strains by hybridization. The resulting hybridization patterns were different for every strain and gave a clear and definite IS fingerprint of each strain. These IS fingerprints can be used to identify and characterize R. meliloti strains rapidly and unequivocally, as they proved to be relatively stable. Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared. This method of IS fingerprinting can also establish whether IS elements are the same, related, or different. Images PMID:1847366

  9. Construction and use of a broad-host-range plasmid expressing the lamB gene for utilization of bacteriophage lambda vectors in the marine bacterium Vibrio harveyi.

    PubMed

    Jasiecki, J; Czy, A; Gabig, M; Wegrzyn, G

    2001-07-01

    The remarkable success of Escherichia coli as a model organism in molecular genetics was dependent, among other things, on its susceptibility to genetic manipulation. Many versatile and sophisticated genetic tools for molecular biology studies are derived from bacteriophage lambda. However, this bacteriophage is specific for E. coli, and thus lambda-based techniques have been restricted to this bacterium. Plasmids expressing the E. coli gene coding for bacteriophage lambda receptor were reported previously, and introduction of such plasmids into cells of some other bacteria made them sensitive to phage lambda infection. However, we found that these systems were not efficient for Vibrio harveyi, one of the most frequently investigated species of marine bacteria. Here we describe construction of a broad-host-range plasmid expressing the lamB gene. Introduction of this plasmid to V. harveyi cells and expression of lamB made this strain susceptible to bacteriophage lambda adsorption and lambda DNA injection. Foreign genetic material could be introduced into cells of this strain using a cosmid vector.

  10. Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection

    PubMed Central

    Taliaferro, Lanyn P.; Galvin, Teresa A.; Ma, Hailun; Shaheduzzaman, Syed; Williams, Dhanya K.; Glasner, Dustin R.; Khan, Arifa S.

    2014-01-01

    Advanced nucleic acid-based technologies are powerful research tools for novel virus discovery but need to be standardized for broader applications such as virus detection in biological products and clinical samples. We have used well-characterized retrovirus stocks to evaluate the limit of detection (LOD) for broad-range PCR with electrospray ionization mass spectrometry (PCR/ESI-MS or PLEX-ID), RT-PCR assays, and virus microarrays. The results indicated that in the absence of background cellular nucleic acids, PLEX-ID and RT-PCR had a similar LOD for xenotropic murine retrovirus-related virus (XMRV; 3.12 particles per µL) whereas sensitivity of virus detection was 10-fold greater using virus microarrays. When virus was spiked into a background of cellular nucleic acids, the LOD using PLEX-ID remained the same, whereas virus detection by RT-PCR was 10-fold less sensitive, and no virus could be detected by microarrays. Expected endogenous retrovirus (ERV) sequences were detected in cell lines tested and known species-specific viral sequences were detected in bovine serum and porcine trypsin. A follow-up strategy was developed using PCR amplification, nucleotide sequencing, and bioinformatics to demonstrate that an RD114-like retrovirus sequence that was detected by PLEX-ID in canine cell lines (Madin-Darby canine kidney (MDCK) and Cf2Th canine thymus) was due to defective, endogenous gammaretrovirus-related sequences. PMID:24777034

  11. Radiometric calibration of optical microscopy and microspectroscopy apparata over a broad spectral range using a special thin-film luminescence standard

    SciTech Connect

    Valenta, J. Greben, M.

    2015-04-15

    Application capabilities of optical microscopes and microspectroscopes can be considerably enhanced by a proper calibration of their spectral sensitivity. We propose and demonstrate a method of relative and absolute calibration of a microspectroscope over an extraordinary broad spectral range covered by two (parallel) detection branches in visible and near-infrared spectral regions. The key point of the absolute calibration of a relative spectral sensitivity is application of the standard sample formed by a thin layer of Si nanocrystals with stable and efficient photoluminescence. The spectral PL quantum yield and the PL spatial distribution of the standard sample must be characterized by separate experiments. The absolutely calibrated microspectroscope enables to characterize spectral photon emittance of a studied object or even its luminescence quantum yield (QY) if additional knowledge about spatial distribution of emission and about excitance is available. Capabilities of the calibrated microspectroscope are demonstrated by measuring external QY of electroluminescence from a standard poly-Si solar-cell and of photoluminescence of Er-doped Si nanocrystals.

  12. Application of a Broad-Range Resequencing Array for Detection of Pathogens in Desert Dust Samples from Kuwait and Iraq ▿

    PubMed Central

    Leski, Tomasz A.; Malanoski, Anthony P.; Gregory, Michael J.; Lin, Baochuan; Stenger, David A.

    2011-01-01

    A significant percentage of the human population is exposed to high levels of naturally occurring airborne dusts. Although the link between airborne particulate inhalation and a variety of respiratory diseases has long been established, little is known about the pathogenic role of the microbial component of the dust. In this study, we applied highly multiplexed PCR and a high-density resequencing microarray (RPM-TEI version 1.0) to screen samples of fine topsoil particles and airborne dust collected in 19 locations in Iraq and Kuwait for the presence of a broad range of human pathogens. The results indicated the presence of potential human pathogens, including Mycobacterium, Brucella, Coxiella burnetii, Clostridium perfringens, and Bacillus. The presence of Coxiella burnetii, a highly infectious potential biowarfare agent, was confirmed and detected in additional samples by use of a more sensitive technique (real-time PCR), indicating a high prevalence of this organism in the analyzed samples. The detection of potentially viable pathogens in breathable dusts from arid regions of Iraq and Kuwait underscores the importance of further study of these environments. PMID:21571877

  13. Highly Active and Robust Metalloporphyrin Catalysts for the Synthesis of Cyclic Carbonates from a Broad Range of Epoxides and Carbon Dioxide.

    PubMed

    Maeda, Chihiro; Shimonishi, Junta; Miyazaki, Ray; Hasegawa, Jun-Ya; Ema, Tadashi

    2016-05-04

    Bifunctional metalloporphyrins with quaternary ammonium bromides (nucleophiles) at the meta, para, or ortho positions of meso-phenyl groups were synthesized as catalysts for the formation of cyclic carbonates from epoxides and carbon dioxide under solvent-free conditions. The meta-substituted catalysts exhibited high catalytic performance, whereas the para- and ortho-substituted catalysts showed moderate and low activity, respectively. DFT calculations revealed the origin of the advantage of the meta-substituted catalyst, which could use the flexible quaternary ammonium cation at the meta position to stabilize various anionic species generated during catalysis. A zinc(II) porphyrin with eight nucleophiles at the meta positions showed very high catalytic activity (turnover number (TON)=240 000 at 120 °C, turnover frequency (TOF)=31 500 h(-1) at 170 °C) at an initial CO2 pressure of 1.7 MPa; catalyzed the reaction even at atmospheric CO2 pressure (balloon) at ambient temperature (20 °C); and was applicable to a broad range of substrates, including terminal and internal epoxides.

  14. Electrospray synthesis of monodisperse polymer particles in a broad (60 nm-2 μm) diameter range: guiding principles and formulation recipes.

    PubMed

    Almería, Begoña; Gomez, Alessandro

    2014-03-01

    This study reports on a methodology to control the size of polymer particles generated by the electrospray (ES) drying route, with emphasis on the generation of biodegradable polymer nanoparticles that are well suited for biomedical applications. The ability to produce spherical poly(lactic-co-glycolic) acid (PLGA) particles with and without encapsulated active agent, with relative standard deviation not exceeding 15%, was demonstrated over a remarkably broad (60 nm-2 μm) diameter range. By judiciously choosing ES parameters and solution properties, we can control the monodispersity and the size of the obtained particles, tailoring it to a specific application. The main parameters affecting particle size include solution electrical conductivity, flow rate and initial polymer volume fraction. Quasi-monodispersity at both the micro- and the more challenging nano-scale was achieved by avoiding Coulomb fission in the spray droplets, via entanglement of the polymer chains within the droplets rather than by charge neutralization. Guiding principles in the formulation of the solutions to satisfy a multiplicity of constraints are provided along with an extensive database of "recipes".

  15. Broad-range (pan) Salmonella and Salmonella serotype typhi-specific real-time PCR assays: potential tools for the clinical microbiologist.

    PubMed

    Farrell, John J; Doyle, Laura J; Addison, Rachel M; Reller, L Barth; Hall, Geraldine S; Procop, Gary W

    2005-03-01

    We describe broad-range salmonellae (ie, Salmonella) and Salmonella serotype Typhi-specific LightCycler (Roche Diagnostics, Indianapolis, IN) real-time polymerase chain reaction assays. We validated these with a battery of 280 bacteria, 108 of which were salmonellae representing 20 serotypes. In addition, 298 isolates from 170 clinical specimens that were suspected to possibly represent Salmonella were tested with the pan- Salmonella assay. Finally, the pan-Salmonella assay also was used to test DNA extracts from 101 archived, frozen stool specimens, 55 of which were culture-positive for salmonellae. Both assays were 100% sensitive and specific when cultured isolates of the battery were tested. The pan- Salmonella assay also characterized correctly all salmonellae on the primary isolation agar and was 96% sensitive (53/55) and 96% specific (49/51) when nucleic acid extracts from direct stool specimens were tested. These assays represent potential tools the clinical microbiologist could use to screen suspect isolates or stool specimens for Salmonella.

  16. Antimicrobial properties of zeolite-X and zeolite-A ion-exchanged with silver, copper, and zinc against a broad range of microorganisms.

    PubMed

    Demirci, Selami; Ustaoğlu, Zeynep; Yılmazer, Gonca Altın; Sahin, Fikrettin; Baç, Nurcan

    2014-02-01

    Zeolites are nanoporous alumina silicates composed of silicon, aluminum, and oxygen in a framework with cations, water within pores. Their cation contents can be exchanged with monovalent or divalent ions. In the present study, the antimicrobial (antibacterial, anticandidal, and antifungal) properties of zeolite type X and A, with different Al/Si ratio, ion exchanged with Ag(+), Zn(2+), and Cu(2+) ions were investigated individually. The study presents the synthesis and manufacture of four different zeolite types characterized by scanning electron microscopy and X-ray diffraction. The ion loading capacity of the zeolites was examined and compared with the antimicrobial characteristics against a broad range of microorganisms including bacteria, yeast, and mold. It was observed that Ag(+) ion-loaded zeolites exhibited more antibacterial activity with respect to other metal ion-embedded zeolite samples. The results clearly support that various synthetic zeolites can be ion exchanged with Ag(+), Zn(2+), and Cu(2+) ions to acquire antimicrobial properties or ion-releasing characteristics to provide prolonged or stronger activity. The current study suggested that zeolite formulations could be combined with various materials used in manufacturing medical devices, surfaces, textiles, or household items where antimicrobial properties are required.

  17. Expanding small-molecule functional metagenomics through parallel screening of broad-host-range cosmid environmental DNA libraries in diverse proteobacteria.

    PubMed

    Craig, Jeffrey W; Chang, Fang-Yuan; Kim, Jeffrey H; Obiajulu, Steven C; Brady, Sean F

    2010-03-01

    The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species.

  18. Expanding Small-Molecule Functional Metagenomics through Parallel Screening of Broad-Host-Range Cosmid Environmental DNA Libraries in Diverse Proteobacteria▿ †

    PubMed Central

    Craig, Jeffrey W.; Chang, Fang-Yuan; Kim, Jeffrey H.; Obiajulu, Steven C.; Brady, Sean F.

    2010-01-01

    The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species. PMID:20081001

  19. Induction of nodule primordia on Phaseolus and Acacia by lipo-chitin oligosaccharide nodulation signals from broad-host-range Rhizobium strain GRH2.

    PubMed

    López-Lara, I M; van der Drift, K M; van Brussel, A A; Haverkamp, J; Lugtenberg, B J; Thomas-Oates, J E; Spaink, H P

    1995-11-01

    Rhizobium wild-type strain GRH2 was originally isolated from the tree, Acacia cyanophylla, and has a broad host-range which includes herbaceous legumes, such as Phaseolus and Trifolium species. Here we show that strains of Rhizobium sp. GRH2, into which heterologous nodD alleles have been introduced, produce a large diversity of both sulphated and non-sulphated lipo-chitin oligosaccharides (LCOs). Most of the molecular species contain an N-methyl group on the reducing-terminal N-acetyl-glucosamine. The LCOs vary in the nature of the fatty acyl chain and in the length of the chitin backbone. The majority of the LCOs have an oligosaccharide chain length of five GlcNAc residues, but a few are oligomers having six GlcNAc units. LCOs purified from GRH2 are able to induce root hair formation and deformation on Acacia cyanophylla and A. melanoxylon plants. We show that an N-vaccenoyl-chitopentaose bearing an N-methyl group is able to induce nodule primordia on Phaseolus vulgaris, A. cyanophylla, and A. melanoxylon, indicating that for these plants an N-methyl modification is sufficient for nodule primordia induction.

  20. Rapid biodegradation of aflatoxin B1 by metabolites of Fusarium sp. WCQ3361 with broad working temperature range and excellent thermostability.

    PubMed

    Wang, Cuiqiong; Li, Zhongyuan; Wang, Hui; Qiu, Haiyan; Zhang, Minghui; Li, Shuang; Luo, Xuegang; Song, Yajian; Zhou, Hao; Ma, Wenjian; Zhang, Tongcun

    2017-03-01

    Contamination of food and feed by aflatoxin B1 (AFB1) poses serious economic and health problems worldwide, so the development of biological methods for effective AFB1 degradation is strongly required. Among three AFB1-degrading microorganisms isolated from moldy peanut, Fusarium sp. WCQ3361 could remove AFB1 extremely effectively, with a degradation ratio of 70.20% after 1 min and 95.38% after 24 h. Its degradation ratio was not much affected by temperature change (0-90 °C) and it also displayed excellent thermostability, maintaining 99.40% residual activity after boiling for 10 min. Since protease K could reduce the AFB1 degradation ratio by 55.15%, it is proposed that the effective component for AFB1 degradation is a protein. The AFB1 degradation ability of Fusarium sp. WCQ3361 was further verified by feed stock detoxification and the MTT test with HepG2 cells. In addition, no degradation products were detected by preliminary liquid chromatography/mass spectrometry, suggesting that AFB1 might be metabolized to products with different chemical characteristics from AFB1. Fusarium sp. WCQ3361 is the first reported AFB1 degradation fungus belonging to the genus Fusarium with broad working temperature range, excellent thermostability and high activity, which provides a potential highly useful solution for dealing with AFB1 contamination in the human diet and animal feed. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  1. Design of thermostable beta-propeller phytases with activity over a broad range of pHs and their overproduction by Pichia pastoris.

    PubMed

    Viader-Salvadó, José M; Gallegos-López, Juan A; Carreón-Treviño, J Gerardo; Castillo-Galván, Miguel; Rojo-Domínguez, Arturo; Guerrero-Olazarán, Martha

    2010-10-01

    Thermostable phytases, which are active over broad pH ranges, may be useful as feed additives, since they can resist the temperatures used in the feed-pelleting process. We designed new beta-propeller phytases, using a structure-guided consensus approach, from a set of amino acid sequences from Bacillus phytases and engineered Pichia pastoris strains to overproduce the enzymes. The recombinant phytases were N-glycosylated, had the correct amino-terminal sequence, showed activity over a pH range of 2.5 to 9, showed a high residual activity after 10 min of heat treatment at 80°C and pH 5.5 or 7.5, and were more thermostable at pH 7.5 than a recombinant form of phytase C from Bacillus subtilis (GenBank accession no. AAC31775). A structural analysis suggested that the higher thermostability may be due to a larger number of hydrogen bonds and to the presence of P257 in a surface loop. In addition, D336 likely plays an important role in the thermostability of the phytases at pH 7.5. The recombinant phytases showed higher thermostability at pH 5.5 than at pH 7.5. This difference was likely due to a different protein total charge at pH 5.5 from that at pH 7.5. The recombinant beta-propeller phytases described here may have potential as feed additives and in the pretreatment of vegetable flours used as ingredients in animal diets.

  2. 6.7-nm Emission from Gd and Tb Plasmas over a Broad Range of Irradiation Parameters Using a Single Laser

    NASA Astrophysics Data System (ADS)

    Yin, Liang; Wang, Hanchen; Reagan, Brendan A.; Baumgarten, Cory; Gullikson, Eric; Berrill, Mark; Shlyaptsev, Vyacheslav N.; Rocca, Jorge J.

    2016-09-01

    We report a comprehensive study of the emission from Gd and Tb laser-produced plasmas in the 6.5-6.7-nm wavelength region for a broad range of laser-irradiation parameters using a single λ =1030 nm laser with tunable pulse duration in the 120-ps-to-4-ns range. The results are of interest for beyond-extreme-ultraviolet (BEUV) lithography of integrated circuits. BEUV emission spectra are measured as a function of laser-pulse duration, emission angle, and spatial location within the plasma. Images of the BEUV-emitting plasma region at the BEUV wavelength are obtained as a function of irradiation parameters. The emission spectrum is observed to broaden and to shift to a longer wavelength as the duration of the driver laser pulses is shortened from nanoseconds to hundreds of picoseconds. Transient self-consistent hydrodynamic and atomic physics simulations show that the picosecond irradiation creates significantly hotter plasmas in which the dominant emission originates from more highly ionized species. Gd-plasma emission driven by nanosecond laser pulses spectrally best matches the responsivity of the energy monitors used, centered near λ =6.74 nm . Spatially resolved spectra of the Gd plasma are acquired for different laser-pulse durations. The conversion efficiency (CE) of Gd /Tb plasma into a 0.6% bandwidth in a 2 π solid angle is determined by integrating angularly resolved measurements obtained using an array of calibrated energy monitors. Similar maximum CEs of about 0.47% for both the Gd and Tb plasmas are obtained. The source size is measured to approximately match the spot size of the laser on target, in agreement with simulations.

  3. microclass: an R-package for 16S taxonomy classification.

    PubMed

    Liland, Kristian Hovde; Vinje, Hilde; Snipen, Lars

    2017-03-16

    Taxonomic classification based on the 16S rRNA gene sequence is important for the profiling of microbial communities. In addition to giving the best possible accuracy, it is also important to quantify uncertainties in the classifications. We present an R package with tools for making such classifications, where the heavy computations are implemented in C++ but operated through the standard R interface. The user may train classifiers based on specialized data sets, but we also supply a ready-to-use function trained on a comprehensive training data set designed specifically for this purpose. This tool also includes some novel ways to quantify uncertainties in the classifications. Based on input sequences of varying length and quality, we demonstrate how the output from the classifications can be used to obtain high quality taxonomic assignments from 16S sequences within the R computing environment. The package is publicly available at the Comprehensive R Archive Network.

  4. Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

    PubMed Central

    Small, Jack; Call, Douglas R.; Brockman, Fred J.; Straub, Timothy M.; Chandler, Darrell P.

    2001-01-01

    We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 μg of total RNA, representing approximately 7.5 × 106 Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR. PMID:11571176

  5. Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes.

    PubMed

    Sessitsch, Angela; Reiter, Birgit; Pfeifer, Ulrike; Wilhelm, Eva

    2002-01-01

    Abstract Endophytic bacteria are ubiquitous in most plants and colonise plants without exhibiting pathogenicity. Studies on the diversity of bacterial endophytes have been mainly approached by characterisation of isolates obtained from internal tissues. Despite the broad application of culture-independent techniques for the analysis of microbial communities in a wide range of natural habitats, little information is available on the species diversity of endophytes. In this study, microbial communities inhabiting stems, roots and tubers of three potato varieties were analysed by 16S rRNA-based techniques such as terminal restriction fragment length polymorphism analysis, denaturing gradient gel electrophoresis as well as 16S rDNA cloning and sequencing. Two individual plant experiments were conducted. In the first experiment plants suffered from light deficiency, whereas healthy and robust plants were obtained in the second experiment. Plants obtained from both experiments showed comparable endophytic populations, but healthy potato plants possessed a significantly higher diversity of endophytes than stressed plants. In addition, plant tissue and variety specific endophytes were detected. Sequence analysis of 16S rRNA genes indicated that a broad phylogenetic spectrum of bacteria is able to colonise plants internally including alpha-, beta-, and gamma-Proteobacteria, high-GC Gram-positives, microbes belonging to the Flexibacter/Cytophaga/Bacteroides group and Planctomycetales. Group-specific analysis of Actinomycetes indicated a higher abundance and diversity of Streptomyces scabiei-related species in the variety Mehlige Mühlviertler, which is known for its resistance against potato common scab caused by S. scabiei.

  6. Seeing red; the development of pON.mCherry, a broad-host range constitutive expression plasmid for Gram-negative bacteria.

    PubMed

    Gebhardt, Michael J; Jacobson, Rachael K; Shuman, Howard A

    2017-01-01

    The development of plasmid-mediated gene expression control in bacteria revolutionized the field of bacteriology. Many of these expression control systems rely on the addition of small molecules, generally metabolites or non-metabolized analogs thereof, to the growth medium to induce expression of the genes of interest. The paradigmatic example of an expression control system is the lac system from Escherichia coli, which typically relies on the Ptac promoter and the Lac repressor, LacI. In many cases, however, constitutive gene expression is desired, and other experimental approaches require the coordinated control of multiple genes. While multiple systems have been developed for use in E. coli and its close relatives, the utility and/or functionality of these tools does not always translate to other species. For example, for the Gram-negative pathogen, Legionella pneumophila, a causative agent of Legionnaires' Disease, the aforementioned Ptac system represents the only well-established expression control system. In order to enhance the tools available to study bacterial gene expression in L. pneumophila, we developed a plasmid, pON.mCherry, which confers constitutive gene expression from a mutagenized LacI binding site. We demonstrate that pON.mCherry neither interferes with other plasmids harboring an intact LacI-Ptac expression system nor alters the growth of Legionella species during intracellular growth. Furthermore, the broad-host range plasmid backbone of pON.mCherry allows constitutive gene expression in a wide variety of Gram-negative bacterial species, making pON.mCherry a useful tool for the greater research community.

  7. Development and validation of broad-range qualitative and clade-specific quantitative molecular probes for assessing mercury methylation in the environment

    SciTech Connect

    Christensen, Geoff A.; Wymore, Ann M.; King, Andrew J.; Podar, Mircea; Hurt, Jr., Richard A.; Santillan, Eugenio U.; Soren, Ally; Brandt, Craig C.; Brown, Steven D.; Palumbo, Anthony V.; Wall, Judy D.; Gilmour, Cynthia C.; Elias, Dwayne A.

    2016-07-15

    Two genes, hgcA and hgcB, are essential for microbial mercury (Hg)-methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability and biogeochemistry is critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea. A distinct PCR product at the expected size was confirmed for all hgcAB+ strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative primers (qPCR) that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88% and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. Here, the resulting data will be essential in developing accurate and robust predictive models of Hg-methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB.

  8. Development and validation of broad-range qualitative and clade-specific quantitative molecular probes for assessing mercury methylation in the environment

    DOE PAGES

    Christensen, Geoff A.; Wymore, Ann M.; King, Andrew J.; ...

    2016-07-15

    Two genes, hgcA and hgcB, are essential for microbial mercury (Hg)-methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability and biogeochemistry is critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea. A distinct PCR product at the expected size was confirmed for all hgcAB+ strains tested via Sanger sequencing.more » Additionally, we developed clade-specific degenerate quantitative primers (qPCR) that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88% and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. Here, the resulting data will be essential in developing accurate and robust predictive models of Hg-methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB.« less

  9. Inactivation of pathogenic viruses by plant-derived tannins: strong effects of extracts from persimmon (Diospyros kaki) on a broad range of viruses.

    PubMed

    Ueda, Kyoko; Kawabata, Ryoko; Irie, Takashi; Nakai, Yoshiaki; Tohya, Yukinobu; Sakaguchi, Takemasa

    2013-01-01

    Tannins, plant-derived polyphenols and other related compounds, have been utilized for a long time in many fields such as the food industry and manufacturing. In this study, we investigated the anti-viral effects of tannins on 12 different viruses including both enveloped viruses (influenza virus H3N2, H5N3, herpes simplex virus-1, vesicular stomatitis virus, Sendai virus and Newcastle disease virus) and non-enveloped viruses (poliovirus, coxsachievirus, adenovirus, rotavirus, feline calicivirus and mouse norovirus). We found that extracts from persimmon (Diospyros kaki), which contains ca. 22% of persimmon tannin, reduced viral infectivity in more than 4-log scale against all of the viruses tested, showing strong anti-viral effects against a broad range of viruses. Other tannins derived from green tea, acacia and gallnuts were effective for some of the viruses, while the coffee extracts were not effective for any of the virus. We then investigated the mechanism of the anti-viral effects of persimmon extracts by using mainly influenza virus. Persimmon extracts were effective within 30 seconds at a concentration of 0.25% and inhibited attachment of the virus to cells. Pretreatment of cells with the persimmon extracts before virus infection or post-treatment after virus infection did not inhibit virus replication. Protein aggregation seems to be a fundamental mechanism underlying the anti-viral effect of persimmon tannin, since viral proteins formed aggregates when purified virions were treated with the persimmon extracts and since the anti-viral effect was competitively inhibited by a non-specific protein, bovine serum albumin. Considering that persimmon tannin is a food supplement, it has a potential to be utilized as a safe and highly effective anti-viral reagent against pathogenic viruses.

  10. Spectral Modulation Effect in Teleseismic P-waves from North Korean Nuclear Tests Recorded in Broad Azimuthal Range and Possible Source Depth Estimation

    NASA Astrophysics Data System (ADS)

    Gitterman, Y.; Kim, S. G.; Hofstetter, R.

    2016-04-01

    Three underground nuclear explosions, conducted by North Korea in 2006, 2009 and 2013, are analyzed. The last two tests were recorded by the Israel Seismic Network. Pronounced coherent minima (spectral nulls) at 1.2-1.3 Hz were revealed in the spectra of teleseismic P -waves. For a ground-truth explosion with a shallow source depth, this phenomenon can be interpreted in terms of the interference between the down-going P-wave and the pP phase reflected from the Earth's surface. This effect was also observed at ISN stations for a Pakistan nuclear explosion at a different frequency 1.7 Hz and the PNE Rubin-2 in West Siberia at 1 Hz, indicating a source-effect and not a site-effect. Similar spectral minima having essentially the same frequency, as at ISN, were observed in teleseismic P-waves for all the three North Korean explosions recorded at networks and arrays in Kazakhstan (KURK), Norway (NNSN), Australia (ASAR, WRA) and Canada (YKA), covering a broad azimuthal range. Data of 2009 and 2013 tests at WRA and KURK arrays showed harmonic spectral modulation with three multiple minima frequencies, evidencing the clear interference effect. These observations support the above-mentioned interpretation. Based on the null frequency dependency on the near-surface acoustic velocity and the source depth, the depth of the North Korean tests was estimated about 2.0-2.1 km. It was shown that the observed null frequencies and the obtained source depth estimates correspond to P- pP interference phenomena in both cases of a vertical shaft or a horizontal drift in a mountain. This unusual depth estimation needs additional validation based on more stations and verification by other methods.

  11. Broad-Range PCR Coupled with Electrospray Ionization Time of Flight Mass Spectrometry for Detection of Bacteremia and Fungemia in Patients with Neutropenic Fever.

    PubMed

    Desmet, S; Maertens, J; Bueselinck, K; Lagrou, K

    2016-10-01

    Infection is an important complication in patients with hematologic malignancies or solid tumors undergoing intensive cytotoxic chemotherapy. In only 20 to 30% of the febrile neutropenic episodes, an infectious agent is detected by conventional cultures. In this prospective study, the performance of broad-range PCR coupled with electrospray ionization time of flight mass spectrometry (PCR/ESI-MS) technology was compared to conventional blood cultures (BC) in a consecutive series of samples from high-risk hematology patients. In 74 patients, BC and a whole-blood sample for PCR/ESI-MS (Iridica BAC BSI; Abbott, Carlsbad, CA, USA) were collected at the start of each febrile neutropenic episode and, in case of persistent fever, also at day 5. During 100 different febrile episodes, 105 blood samples were collected and analyzed by PCR/ESI-MS. There was evidence of a bloodstream infection (BSI) in 36/105 cases (34%), based on 14 cases with both PCR/ESI-MS and BC positivity, 17 cases with BC positivity only, and 5 cases with PCR/ESI-MS positivity only. The sensitivity of PCR/ESI-MS was 45%, specificity was 93%, and the negative predictive value was 80% compared to blood culture. PCR/ESI-MS detected definite pathogens (Fusobacterium nucleatum and Streptococcus pneumoniae) missed by BC, whereas it missed both Gram-negative and Gram-positive organisms detected by BC. PCR/ESI-MS testing detected additional microorganisms but showed a low sensitivity (45%) compared to BC in neutropenic patients. Our results indicate a lower concordance between BC and PCR/ESI-MS in the neutropenic population than what has been previously reported in other patient groups with normal white blood cell distribution, and a lower sensitivity than other PCR-based methods.

  12. Gene regulation on broad host range plasmid RK2: identification of three novel operons whose transcription is repressed by both KorA and KorC.

    PubMed Central

    Thomas, C M; Ibbotson, J P; Wang, N Y; Smith, C A; Tipping, R; Loader, N M

    1988-01-01

    The product of the korA gene of broad host range plasmid RK2 is a key transcriptional repressor which regulates not only the expression of the essential replication gene trfA but also its own expression and that of the kilA operon. It has previously been proposed that korA also encodes a positive activator of transcription of the korC gene, which may act as a transcriptional antiterminator. Here we show that the action of korA in relation to korC can be explained entirely through the korA protein's property as a transcriptional repressor. The limited ability of the previously cloned korC gene to suppress kilC on its own is shown to be due to the fact that korC in RK2 is transcribed from the bla promoter of Tn1 which was deleted in the original korC clones. We demonstrate that korA is a second repressor along with korC of three operons, one of which encodes kilC, the other two not having been described previously and serving an as yet unknown function. We have designated these operons kcrA, B and C for KorC-regulated. Putative kilC is designated kcrC. The homology between the expression signals of these operons suggests that they have arisen by duplication. This is confirmed in the case of kcrA and B by the existence of considerable homology between the products of the first ORFs in each of these operons. Images PMID:2838814

  13. Transcriptome mapping of pAR060302, a blaCMY-2-positive broad-host-range IncA/C plasmid.

    PubMed

    Lang, Kevin S; Danzeisen, Jessica L; Xu, Wayne; Johnson, Timothy J

    2012-05-01

    The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among Escherichia coli and Salmonella enterica strains in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and a propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using E. coli strain DH5α harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure. The effects of ampicillin, florfenicol, or streptomycin exposure were compared to those on cells left untreated at logarithmic phase using Illumina platform-based RNA sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions. A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator. In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance genes floR, bla(CMY-2), aadA, and aacA. Treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics. In contrast, florfenicol treatment resulted in the upregulation of floR and numerous chromosomal genes. Overall, the transcriptome mapping of pAR060302 suggests that it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators.

  14. Seeing red; the development of pON.mCherry, a broad-host range constitutive expression plasmid for Gram-negative bacteria

    PubMed Central

    Jacobson, Rachael K.; Shuman, Howard A.

    2017-01-01

    The development of plasmid-mediated gene expression control in bacteria revolutionized the field of bacteriology. Many of these expression control systems rely on the addition of small molecules, generally metabolites or non-metabolized analogs thereof, to the growth medium to induce expression of the genes of interest. The paradigmatic example of an expression control system is the lac system from Escherichia coli, which typically relies on the Ptac promoter and the Lac repressor, LacI. In many cases, however, constitutive gene expression is desired, and other experimental approaches require the coordinated control of multiple genes. While multiple systems have been developed for use in E. coli and its close relatives, the utility and/or functionality of these tools does not always translate to other species. For example, for the Gram-negative pathogen, Legionella pneumophila, a causative agent of Legionnaires’ Disease, the aforementioned Ptac system represents the only well-established expression control system. In order to enhance the tools available to study bacterial gene expression in L. pneumophila, we developed a plasmid, pON.mCherry, which confers constitutive gene expression from a mutagenized LacI binding site. We demonstrate that pON.mCherry neither interferes with other plasmids harboring an intact LacI-Ptac expression system nor alters the growth of Legionella species during intracellular growth. Furthermore, the broad-host range plasmid backbone of pON.mCherry allows constitutive gene expression in a wide variety of Gram-negative bacterial species, making pON.mCherry a useful tool for the greater research community. PMID:28257493

  15. Broad-Range PCR-Electrospray Ionization Mass Spectrometry for Detection and Typing of Adenovirus and Other Opportunistic Viruses in Stem Cell Transplant Patients

    PubMed Central

    Feghoul, Linda; Mercier-Delarue, Séverine; Dalle, Jean-Hugues; Scieux, Catherine; Chérot, Janine; de Fontbrune, Flore Sicre; Baruchel, André; Socié, Gérard; Simon, François

    2013-01-01

    Hematopoietic stem cell transplant patients are highly susceptible to viral infections. Follow-up after transplantation includes weekly screening using single, virus-specific real-time PCR tests, mainly for viruses in the families Herpesviridae and Adenoviridae that contribute to a high morbidity, especially in pediatric populations. The Abbott PLEX-ID platform combines broad-range PCR with electrospray ionization mass spectrometry to enable the simultaneous detection of multiple pathogens in a single assay. The Viral IC Spectrum assay detects human adenoviruses, viruses from the family Herpesviridae (herpes simplex virus 1 [HSV-1], HSV-2, cytomegalovirus [CMV], Epstein-Barr virus [EBV], varicella-zoster virus [VZV], and human herpesvirus 8 [HHV-8]), human enterovirus, polyomaviruses (BK and JC), and parvovirus B19. We evaluated the performance of the Viral IC Spectrum assay with samples from 16 adult and 36 pediatric stem cell transplant patients. The sensitivity of the Viral IC Spectrum assay compared to real-time PCR quantification using the adenovirus Rgene kit for the detection of adenovirus was 96.7% from plasma samples (n = 92) and 78% from stool samples (n = 100). No adenovirus was detected in samples from noninfected patients (n = 30). PLEX-ID species identification was perfectly concordant with species-specific real-time PCR assays. In plasma and stool samples, the level of amplified products measured by PLEX-ID and the quantity in copies/ml (r = 0.82 and 0.78, respectively) were correlated up to 6 log10 copies/ml. In 67.4% of adenovirus-positive plasma samples, at least one other viral infection was detected; these included BK virus (n = 41), CMV (n = 30), EBV (n = 26), JC virus (n = 9), and HSV-1 (n = 6). The results of this study suggest that the Viral IC Spectrum assay performed on the PLEX-ID platform is reliable for adenovirus infection diagnosis in immunocompromised patients. PMID:24108617

  16. Broad-range PCR-electrospray ionization mass spectrometry for detection and typing of adenovirus and other opportunistic viruses in stem cell transplant patients.

    PubMed

    Legoff, Jérôme; Feghoul, Linda; Mercier-Delarue, Séverine; Dalle, Jean-Hugues; Scieux, Catherine; Chérot, Janine; de Fontbrune, Flore Sicre; Baruchel, André; Socié, Gérard; Simon, François

    2013-12-01

    Hematopoietic stem cell transplant patients are highly susceptible to viral infections. Follow-up after transplantation includes weekly screening using single, virus-specific real-time PCR tests, mainly for viruses in the families Herpesviridae and Adenoviridae that contribute to a high morbidity, especially in pediatric populations. The Abbott PLEX-ID platform combines broad-range PCR with electrospray ionization mass spectrometry to enable the simultaneous detection of multiple pathogens in a single assay. The Viral IC Spectrum assay detects human adenoviruses, viruses from the family Herpesviridae (herpes simplex virus 1 [HSV-1], HSV-2, cytomegalovirus [CMV], Epstein-Barr virus [EBV], varicella-zoster virus [VZV], and human herpesvirus 8 [HHV-8]), human enterovirus, polyomaviruses (BK and JC), and parvovirus B19. We evaluated the performance of the Viral IC Spectrum assay with samples from 16 adult and 36 pediatric stem cell transplant patients. The sensitivity of the Viral IC Spectrum assay compared to real-time PCR quantification using the adenovirus Rgene kit for the detection of adenovirus was 96.7% from plasma samples (n = 92) and 78% from stool samples (n = 100). No adenovirus was detected in samples from noninfected patients (n = 30). PLEX-ID species identification was perfectly concordant with species-specific real-time PCR assays. In plasma and stool samples, the level of amplified products measured by PLEX-ID and the quantity in copies/ml (r = 0.82 and 0.78, respectively) were correlated up to 6 log10 copies/ml. In 67.4% of adenovirus-positive plasma samples, at least one other viral infection was detected; these included BK virus (n = 41), CMV (n = 30), EBV (n = 26), JC virus (n = 9), and HSV-1 (n = 6). The results of this study suggest that the Viral IC Spectrum assay performed on the PLEX-ID platform is reliable for adenovirus infection diagnosis in immunocompromised patients.

  17. Inactivation of Pathogenic Viruses by Plant-Derived Tannins: Strong Effects of Extracts from Persimmon (Diospyros kaki) on a Broad Range of Viruses

    PubMed Central

    Ueda, Kyoko; Kawabata, Ryoko; Irie, Takashi; Nakai, Yoshiaki; Tohya, Yukinobu; Sakaguchi, Takemasa

    2013-01-01

    Tannins, plant-derived polyphenols and other related compounds, have been utilized for a long time in many fields such as the food industry and manufacturing. In this study, we investigated the anti-viral effects of tannins on 12 different viruses including both enveloped viruses (influenza virus H3N2, H5N3, herpes simplex virus-1, vesicular stomatitis virus, Sendai virus and Newcastle disease virus) and non-enveloped viruses (poliovirus, coxsachievirus, adenovirus, rotavirus, feline calicivirus and mouse norovirus). We found that extracts from persimmon (Diospyros kaki), which contains ca. 22% of persimmon tannin, reduced viral infectivity in more than 4-log scale against all of the viruses tested, showing strong anti-viral effects against a broad range of viruses. Other tannins derived from green tea, acacia and gallnuts were effective for some of the viruses, while the coffee extracts were not effective for any of the virus. We then investigated the mechanism of the anti-viral effects of persimmon extracts by using mainly influenza virus. Persimmon extracts were effective within 30 seconds at a concentration of 0.25% and inhibited attachment of the virus to cells. Pretreatment of cells with the persimmon extracts before virus infection or post-treatment after virus infection did not inhibit virus replication. Protein aggregation seems to be a fundamental mechanism underlying the anti-viral effect of persimmon tannin, since viral proteins formed aggregates when purified virions were treated with the persimmon extracts and since the anti-viral effect was competitively inhibited by a non-specific protein, bovine serum albumin. Considering that persimmon tannin is a food supplement, it has a potential to be utilized as a safe and highly effective anti-viral reagent against pathogenic viruses. PMID:23372851

  18. Expression Levels of the Yeast Alcohol Acetyltransferase Genes ATF1, Lg-ATF1, and ATF2 Control the Formation of a Broad Range of Volatile Esters

    PubMed Central

    Verstrepen, Kevin J.; Van Laere, Stijn D. M.; Vanderhaegen, Bart M. P.; Derdelinckx, Guy; Dufour, Jean-Pierre; Pretorius, Isak S.; Winderickx, Joris; Thevelein, Johan M.; Delvaux, Freddy R.

    2003-01-01

    Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1Δ atf2Δ double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes. PMID:12957907

  19. The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family

    PubMed Central

    Li, Xiaobin; Top, Eva M.; Wang, Yafei; Brown, Celeste J.; Yao, Fei; Yang, Shan; Jiang, Yong; Li, Hui

    2015-01-01

    A self-transmissible broad-host-range (BHR) plasmid pSFA231 was isolated from petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs), 29 of which represent genes involved in replication, partitioning and transfer functions of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic analysis shows that pSFA231 shares the common backbone regions with the other PromA plasmids, i.e., genes involved in replication, maintenance and control, and conjugative transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102) and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331), based on the splits network analysis of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98, pIPO2T, and pTer331, suggesting these hypothetical orfs may represent “essential” plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also be accessory genes that were first acquired and then stayed as the plasmid diverged. Our study increases the available collection of complete genome sequences of BHR plasmids, and since pSFA231 is the only characterized PromA plasmid from China, our findings also enhance our understanding of the genetic diversity of this plasmid group in different parts of the world. PMID:25628616

  20. Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; Willson, Richard C.; Fox, George E.

    2002-01-01

    MOTIVATION: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. RESULTS: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.

  1. Phylogenetic relatedness determined between antibiotic resistance and 16S rRNA genes in actinobacteria.

    PubMed

    Sagova-Mareckova, Marketa; Ulanova, Dana; Sanderova, Petra; Omelka, Marek; Kamenik, Zdenek; Olsovska, Jana; Kopecky, Jan

    2015-04-01

    Distribution and evolutionary history of resistance genes in environmental actinobacteria provide information on intensity of antibiosis and evolution of specific secondary metabolic pathways at a given site. To this day, actinobacteria producing biologically active compounds were isolated mostly from soil but only a limited range of soil environments were commonly sampled. Consequently, soil remains an unexplored environment in search for novel producers and related evolutionary questions. Ninety actinobacteria strains isolated at contrasting soil sites were characterized phylogenetically by 16S rRNA gene, for presence of erm and ABC transporter resistance genes and antibiotic production. An analogous analysis was performed in silico with 246 and 31 strains from Integrated Microbial Genomes (JGI_IMG) database selected by the presence of ABC transporter genes and erm genes, respectively. In the isolates, distances of erm gene sequences were significantly correlated to phylogenetic distances based on 16S rRNA genes, while ABC transporter gene distances were not. The phylogenetic distance of isolates was significantly correlated to soil pH and organic matter content of isolation sites. In the analysis of JGI_IMG datasets the correlation between phylogeny of resistance genes and the strain phylogeny based on 16S rRNA genes or five housekeeping genes was observed for both the erm genes and ABC transporter genes in both actinobacteria and streptomycetes. However, in the analysis of sequences from genomes where both resistance genes occurred together the correlation was observed for both ABC transporter and erm genes in actinobacteria but in streptomycetes only in the erm gene. The type of erm resistance gene sequences was influenced by linkage to 16S rRNA gene sequences and site characteristics. The phylogeny of ABC transporter gene was correlated to 16S rRNA genes mainly above the genus level. The results support the concept of new specific secondary metabolite

  2. Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; Willson, Richard C.; Fox, George E.

    2002-01-01

    MOTIVATION: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. RESULTS: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.

  3. A broad-range survey of ticks from livestock in Northern Xinjiang: changes in tick distribution and the isolation of Borrelia burgdorferi sensu stricto.

    PubMed

    Wang, Yuan-Zhi; Mu, Lu-Meng; Zhang, Ke; Yang, Mei-Hua; Zhang, Lin; Du, Jing-Yun; Liu, Zhi-Qiang; Li, Yong-Xiang; Lu, Wei-Hua; Chen, Chuang-Fu; Wang, Yan; Chen, Rong-Gui; Xu, Jun; Yuan, Li; Zhang, Wan-Jiang; Zuo, Wei-Ze; Shao, Ren-Fu

    2015-09-04

    Borreliosis is highly prevalent in Xinjiang Uygur Autonomous Region, China. However, little is known about the presence of Borrelia pathogens in tick species in this region, in addition Borrelia pathogens have not been isolated from domestic animals. We collected adult ticks from domestic animals at 19 sampling sites in 14 counties in northern Xinjiang from 2012 to 2014. Ticks were identified to species by morphology and were molecularly analysed by sequences of mitochondrial 16S rDNA gene; 4-8 ticks of each species at every sampling site were sequenced. 112 live adult ticks were selected for each species in every county, and were used to culture Borrelia pathogens; the genotypes were then determined by sequences of the 5S-23S rRNA intergenic spacer and the outer surface protein A (ospA) gene. A total of 5257 adult ticks, belonging to four genera and seven species, were collected. Compared with three decades ago, the abundance of the five common tick species during the peak ixodid tick season has changed. Certain tick species, such as Rhipicephalus turanicus (Rh. turanicus), was found at Jimusaer, Yining, Fukang, and Chabuchaer Counties for the first time. Additionally, the sequence analyses showed that the Hyalomma asiaticum (Hy. asiaticum), Haemaphysalis punctata (Ha. punctata), and Dermacentor marginatus (D. marginatus) that were collected from different sampling sites (≥3 sites) shared identical 16S rDNA sequences respectively. For the tick species that were collected from the same county, such as Hy. asiaticum from Shihezi County and Rh. turanicus from Yining County, their 16S rDNA sequences showed genetic diversity. In addition, sixteen Borrelia isolates were found in Hy. asiaticum, Ha. punctata, D. marginatus and Rh. turanicus, which infested cattle, sheep, horse and camel in Yining, Chabuchaer, Shihezi and Shawan Counties. All of the isolates were genetically identified as B. Burgdorferi sensu stricto. Warmer and wetter climate may have contributed to the

  4. Different Phenotypes of Walker-Like A Box Mutants of ParA Homolog IncC of Broad-Host-Range IncP Plasmids

    PubMed Central

    Siddique, Azeem; Figurski, David H.

    2012-01-01

    The promiscuous IncPα plasmids RK2 and R995 encode a broad-host-range partition system, whose essential components include the incC and korB genes and a DNA site (OB) to which the korB product binds. IncC2, the smaller of the two incC products, is sufficient for stabilization of R995ΔincC. It is a member of the type Ia ParA family of partition ATPases. To better understand the role of ATP in partition, we constructed three alanine-substitution mutants of IncC2. Each mutation changed a different residue of the Walker-like ATP-binding and hydrolysis motif, including a lysine (K10) conserved solely among members of the ParA and MinD families. All three IncC2 mutants were defective in plasmid partition, but they differed from one another in other respects. The IncC2 T16A mutant, predicted to be defective in Mg2+ coordination, was severely impaired in all activities tested. IncC2 K10A, predicted to be defective in ATP hydrolysis, mediated enhanced incompatibility with R995 derivatives. IncC2 K15A, predicted to be defective in ATP binding, exhibited two distinct incompatibility properties depending on the genotype of the target plasmid. When in trans to plasmids carrying a complementable incC deletion, IncC2 K15A caused dramatic plasmid loss, even at low levels of expression. In trans to wild-type R995 or to R995ΔincC carrying a functional P1 partition system, IncC2 K15A-mediated incompatibility was significantly less than that caused by wild-type IncC2. All three Walker-like A box mutants were also defective for the host toxicity that normally results from co-overexpression of incC and korB. The phenotypes of the mutants support a model in which nucleotide hydrolysis is required for separation of paired plasmid complexes and possible interaction with a host factor. PMID:22579980

  5. The role of flagella and chemotaxis genes in host pathogen interaction of the host adapted Salmonella enterica serovar Dublin compared to the broad host range serovar S. Typhimurium

    PubMed Central

    2013-01-01

    Background The importance of flagella and chemotaxis genes in host pathogen interaction in Salmonella enterica is mainly based on studies of the broad host range serovar, S. Typhimurium, while little is known on the importance in host specific and host adapted serovars, such as S. Dublin. In the current study we have used previously characterized insertion mutants in flagella and chemotaxis genes to investigate this and possible differences in the importance between the two serovars. Results fliC (encoding the structural protein of the flagella) was essential for adhesion and fliC and cheB (CheB restores the chemotaxis system to pre-stimulus conformation) were essential for invasion of S. Dublin into epithelial Int407 cells. In S. Typhimurium, both lack of flagella (fliC/fljB double mutant) and cheB influenced adhesion, and invasion was influenced by lack of both cheA (the histidine-kinase of the chemotaxis system), fliC/fljB and cheB mutation. Uptake in J774A.1 macrophage cells was significantly reduced in cheA, cheB and fliC mutants of S. Dublin, while cheA was dispensable in S. Typhimurium. Removal of flagella in both serotypes caused an increased ability to propagate intracellular in J774 macrophage cells and decreased cytotoxicity toward these cells. Flagella and chemotaxis genes were found not to influence the oxidative response. The induction of IL-6 from J774A-1 cells depended on the presence of flagella in S. Typhimurium, whilst this was not the case following challenge with S. Dublin. Addition of fliC from S. Typhimurium in trans to a fliC mutant of S. Dublin increased cytotoxicity but it did not increase the IL-6 production. Flagella were demonstrated to contribute to the outcome of infection following oral challenge of mice in S. Dublin, while an S. Typhimurium fliC/fljB mutant showed increased virulence following intra peritoneal challenge. Conclusions The results showed that flagella and chemotaxis genes differed in their role in host pathogen

  6. Equivalent asthma control and systemic safety of inhaled budesonide delivered via HFA-134a or CFC propellant in a broad range of doses.

    PubMed

    Grzelewska-Rzymowska, I; Malolepszy, J; de Molina, M; Sladek, K; Zarkovice, J; Siergiejko, Z

    2003-11-01

    was also noted with both propellants. In conclusion, the budesonide HFA-134a formulation given with a spacer device provided an equivalent asthma control with that of a corresponding CFC product, when administered in stable patients treated with inhaled corticosteroids in a broad range of daily doses. The use of the new propellant did not modify the safety profile of inhaled budesonide.

  7. Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment

    PubMed Central

    Christensen, Geoff A.; Wymore, Ann M.; King, Andrew J.; Podar, Mircea; Hurt, Richard A.; Santillan, Eugenio U.; Soren, Ally; Brandt, Craig C.; Brown, Steven D.; Palumbo, Anthony V.; Wall, Judy D.; Gilmour, Cynthia C.

    2016-01-01

    ABSTRACT Two genes, hgcA and hgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea genomes. A distinct PCR product at the expected size was confirmed for all hgcAB+ strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88%, and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes, and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB. IMPORTANCE The neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The

  8. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys.

    PubMed

    Walters, William; Hyde, Embriette R; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A; Jansson, Janet K; Caporaso, J Gregory; Fuhrman, Jed A; Apprill, Amy; Knight, Rob

    2016-01-01

    Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5' end, allowing for a range of different 3' primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for

  9. Fully stereocontrolled total syntheses of the prostacyclin analogues 16S-iloprost and 16S-3-oxa-iloprost by a common route, using alkenylcopper-azoalkene conjugate addition, asymmetric olefination, and allylic alkylation.

    PubMed

    Kramp, Guido J; Kim, Mikhail; Gais, Hans-Joachim; Vermeeren, Cornelia

    2005-12-21

    In this article we describe fully stereocontrolled total syntheses of 16S-iloprost (16S-2), the most active component of the drugs Ilomedin and Ventavis, and of 16S-3-oxa-iloprost (16S-3), a close analogue of 16S-2 having the potential for a high oral activity, by a new and common route. The key steps of this route are (1) the establishment of the complete C13-C20 omega side chain of the target molecules through a stereoselective conjugate addition of the alkenylcopper derivative 9 to the bicyclic C6-C12 azoalkene 10 with formation of hydrazone 8, (2) the diastereoselective olefination of ketone 7 with the chiral phosphoryl acetate 39, and (3) the regio- and stereoselective alkylation of the allylic acetate 43 with cuprate 42. These measures allowed the 5E,15S,16S-stereoselective synthesis of 16S-2 and 16S-3, a goal which had previously not been achieved. Azoalkene 10 was obtained from the achiral bicyclic C6-C12 ketone 11 as previously described by using as key step an enantioselective deprotonation. The configuration at C16 of omega-side chain building block 9 has been installed with high stereoselectivity by the oxazolidinone method and that at C15 by a diastereoselective oxazaborolidine-catalyzed reduction of the C13-C20 ketone 23 with catecholborane. Surprisingly, a high diastereoselectivity in the reduction of 23 was only obtained by using 2 equiv of oxazaborolidine 24. Application of substoichiometric amounts of 24 resulted in irreproducible diastereoselectivities ranging from very high to nil.

  10. Further development of the unified multiscale Eulerian model for a broad range of spatial and temporal scales within the new National Environmental Modeling System

    NASA Astrophysics Data System (ADS)

    Janjic, Z.

    2009-04-01

    A unified Nonhydrostatic Multiscale Model on the Arakawa B grid (NMMB) designed for a broad range of spatial and temporal scales has been under development within the Earth System Modeling Framework (ESMF) at the National Centers for Environmental Prediction (NCEP) as a part of the new National Environmental Modeling System (NEMS). The model follows the general modeling philosophy of the NCEP's WRF NMM grid-point regional dynamical core. The model uses the regular latitude-longitude grid for the global domain, and a rotated latitude-longitude grid in regional applications. The nonhydrostatic component of the model dynamics is introduced through an add-on module that can be turned on or off depending on resolution. The "isotropic" quadratic conservative finite-volume horizontal differencing employed in the model conserves a variety of basic and derived dynamical and quadratic quantities and preserves some important properties of differential operators. Among these, the conservation of energy and enstrophy improves the accuracy of the nonlinear dynamics of the model on all scales. "Across the pole" polar boundary conditions are specified in the global limit and the polar filter selectively slows down the wave components of the basic dynamical variables that would otherwise propagate faster in the zonal direction than the fastest wave propagating in the meridional direction. Several upgrades have been recently incorporated into the model. As a compromise between requirements for affordability and accuracy, a fast Eulerian conservative and positive definite scheme has been developed for model tracers. Conservative monotonization is applied in order to control over-steepening within the tracer advection scheme. The tests performed so far have been encouraging concerning the tracer mass conservation and shape preservation, as well as computational efficiency. As an effort for unification among NEMS models that was expected to improve data assimilation, several algorithms

  11. Universal bacterial identification by mass spectrometry of 16S ribosomal RNA cleavage products

    NASA Astrophysics Data System (ADS)

    Jackson, George W.; McNichols, Roger J.; Fox, George E.; Willson, Richard C.

    2007-03-01

    The public availability of over 180,000 bacterial 16S ribosomal RNA (rRNA) sequences has facilitated microbial identification and classification using nucleic acid hybridization and other molecular approaches. Species-specific PCR, microarrays, and in situ hybridization are based on the presence of unique subsequences in the target sequence and therefore require prior knowledge of what organisms are likely to be present in a sample. Mass spectrometry is not limited by a pre-synthesized inventory of probe/primer sequences. It has already been demonstrated that organism identification can be recovered from mass spectra using various methods including base-specific cleavage of nucleic acids. The feasibility of broad bacterial identification by comparing such mass spectral patterns to predictive databases derived from virtually all previously sequenced strains has yet to be demonstrated, however. Herein, we present universal bacterial identification by base-specific cleavage, mass spectrometry, and an efficient coincidence function for rapid spectral scoring against a large database of predicted "mass catalogs". Using this approach in conjunction with universal PCR of the 16S rDNA gene, four bacterial isolates and an uncultured clone were successfully identified against a database of predicted cleavage products derived 6rom over 47,000 16S rRNA sequences representing all major bacterial taxaE At present, the conventional DNA isolation and PCR steps require approximately 2 h, while subsequent transcription, enzymatic cleavage, mass spectrometric analysis, and database comparison require less than 45 min. All steps are amenable to high-throughput implementation.

  12. Comparative Evaluation of Four Bacteria-Specific Primer Pairs for 16S rRNA Gene Surveys

    PubMed Central

    Thijs, Sofie; Op De Beeck, Michiel; Beckers, Bram; Truyens, Sascha; Stevens, Vincent; Van Hamme, Jonathan D.; Weyens, Nele; Vangronsveld, Jaco

    2017-01-01

    Bacterial taxonomic community analyses using PCR-amplification of the 16S rRNA gene and high-throughput sequencing has become a cornerstone in microbiology research. To reliably detect the members, or operational taxonomic units (OTUs), that make up bacterial communities, taxonomic surveys rely on the use of the most informative PCR primers to amplify the broad range of phylotypes present in up-to-date reference databases. However, primers specific for the domain Bacteria were often developed some time ago against database versions that are now out of date. Here we evaluated the performance of four bacterial primers for characterizing complex microbial communities in explosives contaminated and non-contaminated forest soil and by in silico evaluation against the current SILVA123 database. Primer pair 341f/785r produced the highest number of bacterial OTUs, phylogenetic richness, Shannon diversity, low non-specificity and most reproducible results, followed by 967f/1391r and 799f/1193r. Primer pair 68f/518r showed overall low coverage and a bias toward Alphaproteobacteria. In silico, primer pair 341f/785r showed the highest coverage of the domain Bacteria (96.1%) with no obvious bias toward the majority of bacterial species. This suggests the high utility of primer pair 341f/785r for soil and plant-associated bacterial microbiome studies. PMID:28400755

  13. Nested polymerase chain reaction (PCR) targeting 16S rDNA for bacterial identification in empyema.

    PubMed

    Prasad, Rajniti; Kumari, Chhaya; Das, B K; Nath, Gopal

    2014-05-01

    Empyema in children causes significant morbidity and mortality. However, identification of organisms is a major concern. To detect bacterial pathogens in pus specimens of children with empyema by 16S rDNA nested polymerase chain reaction (PCR) and correlate it with culture and sensitivity. Sixty-six children admitted to the paediatric ward with a diagnosis of empyema were enrolled prospectively. Aspirated pus was subjected to cytochemical examination, culture and sensitivity, and nested PCR targeting 16S rDNA using a universal eubacterial primer. Mean (SD) age was 5·8 (1·8) years (range 1-13). Analysis of aspirated pus demonstrated total leucocyte count >1000×10(6)/L, elevated protein (≧20 g/L) and decreased glucose (≤2·2 mmol/L) in 80·3%, 98·5% and 100%, respectively. Gram-positive cocci were detected in 29 (43·9%) and Gram-negative bacilli in two patients. Nested PCR for the presence of bacterial pathogens was positive in 50·0%, compared with 36·3% for culture. 16S rDNA PCR improves rates of detection of bacteria in pleural fluid, and can detect bacterial species in a single assay as well as identifying unusual and unexpected causal agents.

  14. Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification.

    PubMed

    Wagner, Josef; Coupland, Paul; Browne, Hilary P; Lawley, Trevor D; Francis, Suzanna C; Parkhill, Julian

    2016-11-14

    Bio full-length bacterial 16S rRNA gene datasets generated 261 OTUs, which were grouped into 52 species, of which 54% were shared with the MiSeq dataset. Alpha diversity index reported a higher diversity in the MiSeq dataset. The PacBio sequencing error rate is now in the same range of the previously widely used Roche 454 sequencing platform and current MiSeq platform. Species-level microbiome analysis revealed some inconsistencies between the full-length bacterial 16S rRNA gene capillary sequencing and PacBio sequencing.

  15. [PCR rDNA 16S used for the etiological diagnosis of blood culture negative endocarditis].

    PubMed

    Baty, G; Lanotte, P; Hocqueloux, L; Prazuck, T; Bret, L; Romano, M; Mereghetti, L

    2010-06-01

    We report the case of a 55 year-old man presenting with a double aortic and mitral endocarditis for which resected valve culture was repeatedly negative. Specific PCR made on valves because of highly positive blood tests for Bartonella henselae remained negative. A molecular approach was made with 16S rDNA PCR, followed by sequencing. Bartonella quintana was identified as the etiology of endocarditis. B. quintana, "fastidious" bacteria, even if hard to identify in a laboratory, is often reported as a blood culture negative endocarditis (BCNE) agent. Molecular biology methods have strongly improved the diagnosis of BCNE. We propose a review of the literature focusing on the interest of broad-spectrum PCR on valve for the etiological diagnosis of BCNE.

  16. Characteristic archaebacterial 16S rRNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    McGill, T. J.; Jurka, J.; Sobieski, J. M.; Pickett, M. H.; Woese, C. R.; Fox, G. E.

    1986-01-01

    A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

  17. Characteristic archaebacterial 16S rRNA oligonucleotides.

    PubMed

    McGill, T J; Jurka, J; Sobieski, J M; Pickett, M H; Woese, C R; Fox, G E

    1986-01-01

    A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

  18. Influenza B-cells protective epitope characterization: a passkey for the rational design of new broad-range anti-influenza vaccines.

    PubMed

    Clementi, Nicola; Criscuolo, Elena; Castelli, Matteo; Mancini, Nicasio; Clementi, Massimo; Burioni, Roberto

    2012-11-14

    The emergence of new influenza strains causing pandemics represents a serious threat to human health. From 1918, four influenza pandemics occurred, caused by H1N1, H2N2 and H3N2 subtypes. Moreover, in 1997 a novel influenza avian strain belonging to the H5N1 subtype infected humans. Nowadays, even if its transmission is still circumscribed to avian species, the capability of the virus to infect humans directly from avian reservoirs can result in fatalities. Moreover, the risk that this or novel avian strains could adapt to inter-human transmission, the development of resistance to anti-viral drugs and the lack of an effective prevention are all incumbent problems for the world population. In this scenario, the identification of broadly neutralizing monoclonal antibodies (mAbs) directed against conserved regions shared among influenza isolates has raised hopes for the development of monoclonal antibody-based immunotherapy and "universal" anti-influenza vaccines.

  19. Technologically important extremophile 16S rRNA sequence Shannon entropy and fractal property comparison with long term dormant microbes

    NASA Astrophysics Data System (ADS)

    Holden, Todd; Gadura, N.; Dehipawala, S.; Cheung, E.; Tuffour, M.; Schneider, P.; Tremberger, G., Jr.; Lieberman, D.; Cheung, T.

    2011-10-01

    Technologically important extremophiles including oil eating microbes, uranium and rocket fuel perchlorate reduction microbes, electron producing microbes and electrode electrons feeding microbes were compared in terms of their 16S rRNA sequences, a standard targeted sequence in comparative phylogeny studies. Microbes that were reported to have survived a prolonged dormant duration were also studied. Examples included the recently discovered microbe that survives after 34,000 years in a salty environment while feeding off organic compounds from other trapped dead microbes. Shannon entropy of the 16S rRNA nucleotide composition and fractal dimension of the nucleotide sequence in terms of its atomic number fluctuation analyses suggest a selected range for these extremophiles as compared to other microbes; consistent with the experience of relatively mild evolutionary pressure. However, most of the microbes that have been reported to survive in prolonged dormant duration carry sequences with fractal dimension between 1.995 and 2.005 (N = 10 out of 13). Similar results are observed for halophiles, red-shifted chlorophyll and radiation resistant microbes. The results suggest that prolonged dormant duration, in analogous to high salty or radiation environment, would select high fractal 16S rRNA sequences. Path analysis in structural equation modeling supports a causal relation between entropy and fractal dimension for the studied 16S rRNA sequences (N = 7). Candidate choices for high fractal 16S rRNA microbes could offer protection for prolonged spaceflights. BioBrick gene network manipulation could include extremophile 16S rRNA sequences in synthetic biology and shed more light on exobiology and future colonization in shielded spaceflights. Whether the high fractal 16S rRNA sequences contain an asteroidlike extra-terrestrial source could be speculative but interesting.

  20. Rapid identification of bovine mastitis pathogens by high-resolution melt analysis of 16S rDNA sequences.

    PubMed

    Ajitkumar, Praseeda; Barkema, Herman W; De Buck, Jeroen

    2012-03-23

    Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with

  1. White LEDs as broad spectrum light sources for spectrophotometry: demonstration in the visible spectrum range in a diode-array spectrophotometric detector.

    PubMed

    Piasecki, Tomasz; Breadmore, Michael C; Macka, Mirek

    2010-11-01

    Although traditional lamps, such as deuterium lamps, are suitable for bench-top instrumentation, their compatibility with the requirements of modern miniaturized instrumentation is limited. This study investigates the option of utilizing solid-state light source technology, namely white LEDs, as a broad band spectrum source for spectrophotometry. Several white light LEDs of both RGB and white phosphorus have been characterized in terms of their emission spectra and energy output and a white phosphorus Luxeon LED was then chosen for demonstration as a light source for visible-spectrum spectrophotometry conducted in CE. The Luxeon LED was fixed onto the base of a dismounted deuterium (D(2) ) lamp so that the light-emitting spot was geometrically positioned exactly where the light-emitting spot of the original D(2) lamp is placed. In this manner, the detector of a commercial CE instrument equipped with a DAD was not modified in any way. As the detector hardware and electronics remained the same, the change of the deuterium lamp for the Luxeon white LED allowed a direct comparison of their performances. Several anionic dyes as model analytes with absorption maxima between 450 and 600 nm were separated by CE in an electrolyte of 0.01 mol/L sodium tetraborate. The absorbance baseline noise as the key parameter was 5 × lower for the white LED lamp, showing clearly superior performance to the deuterium lamp in the available, i.e. visible part of the spectrum.

  2. A New Broad Range Plasmid for DNA Delivery in Eukaryotic Cells Using Lactic Acid Bacteria: In Vitro and In Vivo Assays.

    PubMed

    Mancha-Agresti, Pamela; Drumond, Mariana Martins; Carmo, Fillipe Luiz Rosa do; Santos, Monica Morais; Santos, Janete Soares Coelho Dos; Venanzi, Franco; Chatel, Jean-Marc; Leclercq, Sophie Yvette; Azevedo, Vasco

    2017-03-17

    Lactococcus lactis is well documented as a promising candidate for development of novel oral live vaccines. It has been broadly engineered for heterologous expression, as well as for plasmid expression vector delivery, directly inside eukaryotic cells, for DNA vaccine, or as therapeutic vehicle. This work describes the characteristics of a new plasmid, pExu (extra chromosomal unit), for DNA delivery using L. lactis and evaluates its functionality both by in vitro and in vivo assays. This plasmid exhibits the following features: (1) a theta origin of replication and (2) an expression cassette containing a multiple cloning site and a eukaryotic promoter, the cytomegalovirus (pCMV). The functionality of pExu:egfp was evaluated by fluorescence microscopy. The L. lactis MG1363 (pExu:egfp) strains were administered by gavage to Balb/C mice and the eGFP expression was monitored by fluorescence microscopy. The pExu vector has demonstrated an excellent stability either in L. lactis or in Escherichia coli. The eGFP expression at different times in in vitro assay showed that 15.8% of CHO cells were able to express the protein after transfection. The enterocytes of mice showed the expression of eGFP protein. Thus, L. lactis carrying the pExu is a good candidate to deliver genes into eukaryotic cells.

  3. Heterologous production of an acidic thermostable lipase with broad-range pH activity from thermophilic fungus Neosartorya fischeri P1.

    PubMed

    Sun, Qiaoqiao; Wang, Hui; Zhang, Huitu; Luo, Huiying; Shi, Pengjun; Bai, Yingguo; Lu, Fuping; Yao, Bin; Huang, Huoqing

    2016-11-01

    Thermophilic Neosartorya fischeri P1 is an excellent lipase producer and harbors seven lipase genes. All genes were found to be functional after heterologous expression in Escherichia coli. One of them, LIP09, showed high-level expression in Pichia pastoris with the yield of 2.0 g/L in a 3.7-L fermentor. Deduced amino acid sequence of LIP09 consists of a putative signal peptide (residues 1-19) and a mature polypeptide (residues 20-562). Compared with other fungal counterparts, purified recombinant LIP09 has some superior properties. It exhibited maximum activity at 60°C and pH 5.0, had broad pH adaptability (>60% activity at pH 3.5-8.0) and stability (retaining >90% activity after incubation at pH 3.0-7.0 for 1 h at 40°C), and was highly thermostable (retaining >96% activity after incubation at 50°C for 30 min). The r-LIP09 had a preference for the medium-chain length p-nitrophenyl esters (C12) rather than short and long-chain length substrates. The high-level expression and excellent properties make LIP09 a potential enzyme candidate in food and feed industries. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  4. Identification of non-tuberculous mycobacteria: utility of the GenoType Mycobacterium CM/AS assay compared with HPLC and 16S rRNA gene sequencing.

    PubMed

    Lee, Andie S; Jelfs, Peter; Sintchenko, Vitali; Gilbert, Gwendolyn L

    2009-07-01

    Non-tuberculous mycobacteria (NTM) causing clinical disease have become increasingly common and more diverse. A new reverse line probe assay, GenoType Mycobacterium CM/AS (Hain Lifescience), was evaluated for identification of a broad range of NTM. It was compared with phenotypic (HPLC) and molecular (DNA probes, in-house real-time multiplex species-specific PCR, 16S rRNA gene PCR and sequencing) identification techniques, which together provided the reference 'gold standard'. A total of 131 clinical isolates belonging to 31 Mycobacterium species and 19 controls, including 5 non-Mycobacterium species, was used. Concordant results between the GenoType Mycobacterium assay and the reference identification were obtained in 119/131 clinical isolates (90.8 %). Identification of Mycobacterium abscessus and Mycobacterium lentiflavum by the assay was problematic. The GenoType Mycobacterium assay enables rapid identification of a broad range of potentially clinically significant Mycobacterium species, but some species require further testing to differentiate or confirm ambiguous results.

  5. Broad-host-range plasmids for red fluorescent protein labeling of gram-negative bacteria for use in the zebrafish model system.

    PubMed

    Singer, John T; Phennicie, Ryan T; Sullivan, Matthew J; Porter, Laura A; Shaffer, Valerie J; Kim, Carol H

    2010-06-01

    To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-beta-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI(q) carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen.

  6. Evaluation of the Broad-Range PCR/ESI-MS Technology in Blood Specimens for the Molecular Diagnosis of Bloodstream Infections.

    PubMed

    Jordana-Lluch, Elena; Giménez, Montserrat; Quesada, M Dolores; Rivaya, Belén; Marcó, Clara; Domínguez, M Jesús; Arméstar, Fernando; Martró, Elisa; Ausina, Vicente

    2015-01-01

    Rapid identification of the etiological agent in bloodstream infections is of vital importance for the early administration of the most appropriate antibiotic therapy. Molecular methods may offer an advantage to current culture-based microbiological diagnosis. The goal of this study was to evaluate the performance of IRIDICA, a platform based on universal genetic amplification followed by mass spectrometry (PCR/ESI-MS) for the molecular diagnosis of sepsis-related pathogens directly from the patient's blood. A total of 410 whole blood specimens from patients admitted to Emergency Room (ER) and Intensive Care Unit (ICU) with clinical suspicion of sepsis were tested with the IRIDICA BAC BSI Assay (broad identification of bacteria and Candida spp.). Microorganisms grown in culture and detected by IRIDICA were compared considering blood culture as gold standard. When discrepancies were found, clinical records and results from other cultures were taken into consideration (clinical infection criterion). The overall positive and negative agreement of IRIDICA with blood culture in the analysis by specimen was 74.8% and 78.6%, respectively, rising to 76.9% and 87.2% respectively, when compared with the clinical infection criterion. Interestingly, IRIDICA detected 41 clinically significant microorganisms missed by culture, most of them from patients under antimicrobial treatment. Of special interest were the detections of one Mycoplasma hominis and two Mycobacterium simiae in immunocompromised patients. When ICU patients were analyzed separately, sensitivity, specificity, positive and negative predictive values compared with blood culture were 83.3%, 78.6%, 33.9% and 97.3% respectively, and 90.5%, 87.2%, 64.4% and 97.3% respectively, in comparison with the clinical infection criterion. IRIDICA is a promising technology that offers an early and reliable identification of a wide variety of pathogens directly from the patient's blood within 6h, which brings the opportunity to

  7. Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C.

    PubMed

    Strokappe, Nika; Szynol, Agnieszka; Aasa-Chapman, Marlèn; Gorlani, Andrea; Forsman Quigley, Anna; Hulsik, David Lutje; Chen, Lei; Weiss, Robin; de Haard, Hans; Verrips, Theo

    2012-01-01

    Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120(Ds2)), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B'/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides.

  8. Leuconostoc pseudomesenteroides WCFur3 partial 16S rRNA gene

    USDA-ARS?s Scientific Manuscript database

    This study used a partial 535 base pair 16S rRNA gene sequence to identify a bacterial isolate. Fatty acid profiles are consistent with the 16S rRNA gene sequence identification of this bacterium. The isolate was obtained from a compost bin in Fort Collins, Colorado, USA. The 16S rRNA gene sequen...

  9. 16S Classifier: A Tool for Fast and Accurate Taxonomic Classification of 16S rRNA Hypervariable Regions in Metagenomic Datasets

    PubMed Central

    Chaudhary, Nikhil; Sharma, Ashok K.; Agarwal, Piyush; Gupta, Ankit; Sharma, Vineet K.

    2015-01-01

    The diversity of microbial species in a metagenomic study is commonly assessed using 16S rRNA gene sequencing. With the rapid developments in genome sequencing technologies, the focus has shifted towards the sequencing of hypervariable regions of 16S rRNA gene instead of full length gene sequencing. Therefore, 16S Classifier is developed using a machine learning method, Random Forest, for faster and accurate taxonomic classification of short hypervariable regions of 16S rRNA sequence. It displayed precision values of up to 0.91 on training datasets and the precision values of up to 0.98 on the test dataset. On real metagenomic datasets, it showed up to 99.7% accuracy at the phylum level and up to 99.0% accuracy at the genus level. 16S Classifier is available freely at http://metagenomics.iiserb.ac.in/16Sclassifier and http://metabiosys.iiserb.ac.in/16Sclassifier. PMID:25646627

  10. BOLD fMRI and Somatosensory Evoked Potentials Are Well Correlated Over a Broad Range of Frequency Content of Somatosensory Stimulation of the Rat Forepaw

    PubMed Central

    Goloshevsky, Artem G.; Silva, Afonso C.; Dodd, Stephen J.; Koretsky, Alan P.

    2008-01-01

    Electrical stimulation of the rat paw is commonly used to study the hemodynamic, metabolic, and neuronal mechanisms of functional MRI (fMRI) responses in somatosensory cortex. Several groups have reported good correlation between the Blood Oxygenation Level Dependent (BOLD) fMRI signal and somatosensory evoked potentials (SEPs) using short, typically 300 μs, square stimulation pulses. The spectral power of these short pulses is evenly distributed over a wide range of frequencies and thus the effects of the frequency content of the stimulation pulse on fMRI responses have not been previously described. Here, the effects that different stimulation pulse waveforms with a range of frequency content have on neuronal activity, as measured by SEPs, and on the amplitude of the BOLD fMRI signal in rat somatosensory cortex are investigated. The peak-to-peak SEP amplitudes increased as the power in the high frequency harmonics of the different pulse waveforms increased, using either triangular or sinusoidal stimuli waveforms from 9 Hz to 180 Hz. Similarly, BOLD fMRI response increased with increased high frequency content of the stimulation pulse. There was a linear correlation between SEPs and BOLD fMRI over the full range of frequency content in the stimulations. PMID:18206862

  11. Evaluation of the Broad-Range PCR/ESI-MS Technology in Blood Specimens for the Molecular Diagnosis of Bloodstream Infections

    PubMed Central

    Jordana-Lluch, Elena; Giménez, Montserrat; Quesada, Mª Dolores; Rivaya, Belén; Marcó, Clara; Domínguez, Mª Jesús; Arméstar, Fernando; Martró, Elisa; Ausina, Vicente

    2015-01-01

    Background Rapid identification of the etiological agent in bloodstream infections is of vital importance for the early administration of the most appropriate antibiotic therapy. Molecular methods may offer an advantage to current culture-based microbiological diagnosis. The goal of this study was to evaluate the performance of IRIDICA, a platform based on universal genetic amplification followed by mass spectrometry (PCR/ESI-MS) for the molecular diagnosis of sepsis-related pathogens directly from the patient’s blood. Methods A total of 410 whole blood specimens from patients admitted to Emergency Room (ER) and Intensive Care Unit (ICU) with clinical suspicion of sepsis were tested with the IRIDICA BAC BSI Assay (broad identification of bacteria and Candida spp.). Microorganisms grown in culture and detected by IRIDICA were compared considering blood culture as gold standard. When discrepancies were found, clinical records and results from other cultures were taken into consideration (clinical infection criterion). Results The overall positive and negative agreement of IRIDICA with blood culture in the analysis by specimen was 74.8% and 78.6%, respectively, rising to 76.9% and 87.2% respectively, when compared with the clinical infection criterion. Interestingly, IRIDICA detected 41 clinically significant microorganisms missed by culture, most of them from patients under antimicrobial treatment. Of special interest were the detections of one Mycoplasma hominis and two Mycobacterium simiae in immunocompromised patients. When ICU patients were analyzed separately, sensitivity, specificity, positive and negative predictive values compared with blood culture were 83.3%, 78.6%, 33.9% and 97.3% respectively, and 90.5%, 87.2%, 64.4% and 97.3% respectively, in comparison with the clinical infection criterion. Conclusions IRIDICA is a promising technology that offers an early and reliable identification of a wide variety of pathogens directly from the patient’s blood

  12. A paralogue of the phosphomutase-like gene family in Candida glabrata, CgPmu2, gained broad-range phosphatase activity due to a small number of clustered substitutions.

    PubMed

    Orlando, Kelly A; Iosue, Christine L; Leone, Sarah G; Davies, Danielle L; Wykoff, Dennis D

    2015-10-15

    Inorganic phosphate is required for a range of cellular processes, such as DNA/RNA synthesis and intracellular signalling. The phosphate starvation-inducible phosphatase activity of Candida glabrata is encoded by the gene CgPMU2 (C. glabrata phosphomutase-like protein). CgPMU2 is part of a three-gene family (∼75% identical) created through gene duplication in the C. glabrata clade; only CgPmu2 is a PHO-regulated broad range acid phosphatase. We identified amino acids that confer broad range phosphatase activity on CgPmu2 by creating fusions of sections of CgPMU2 with CgPMU1, a paralogue with little broad range phosphatase activity. We used site-directed mutagenesis on various fusions to sequentially convert CgPmu1 to CgPmu2. Based on molecular modelling of the Pmu proteins on to a histidine phosphatase crystal structure, clusters of amino acids were found in two distinct regions that were able to confer phosphatase activity. Substitutions in these two regions together conferred broad phosphatase activity on CgPmu1. Interestingly, one change is a histidine adjacent to the active site histidine of CgPmu2 and it exhibits a novel ability to partially replace the conserved active site histidine in CgPmu2. Additionally, a second amino acid change was able to confer nt phosphatase activity to CgPmu1, suggesting single amino acid changes neofunctionalize CgPmu2. © 2015 Authors; published by Portland Press Limited.

  13. Liquid-phase epitaxy grown PbSnTe distributed feedback laser diodes with broad continuous single-mode tuning range

    NASA Technical Reports Server (NTRS)

    Hsieh, H.-H.; Fonstad, C. G.

    1980-01-01

    Distributed feedback (DFB) pulsed laser operation has been demonstrated in stripe geometry Pb(1-x)Sn(x)Te double-heterostructures grown by liquid-phase epitaxy. The grating structure of 0.79 micron periodicity operates in first order near 12.8 microns and was fabricated prior to the liquid-phase epitaxial growth using holographic exposure techniques. These DFB lasers had moderate thresholds, 3.6 kA/sq cm, and the output power versus current curves exhibited a sharp turn-on free of kinks. Clean, single-mode emission spectra, continuously tunable over a range in excess of 20 per cm, centered about 780 per cm (12.8 microns), and at an average rate of 1.2 per cm-K from 9 to 26 K, were observed. While weaker modes could at times be seen in the spectrum, substantially single-mode operation was obtained over the entire operating range and to over 10 times threshold.

  14. Influence of CO molecular impurity on the structural and thermodynamic properties of fullerite C60, in a broad range of sorption temperatures

    NASA Astrophysics Data System (ADS)

    Meleshko, V. V.; Legchenkova, I. V.; Stetsenko, Y. E.; Prokhvatilov, A. I.

    2016-02-01

    An x-ray diffraction study of how sorption of CO gas at a pressure of 30 atm in the temperature range of 150-600 °C influences the structural characteristics of polycrystalline and single crystal fullerite C60. The sorption kinetics are studied by constructing a dependence of the lattice parameter on the time it takes for fullerite to be saturated by CO molecules. At temperatures Tsorb > 300 °C there is an observed dissociation of carbon monoxide, accompanied by the precipitation of carbon powder and the chemical interaction of atomic oxygen with C60 and CO molecules, and possibly with the carbon condensate. These processes have a strong influence on the structural characteristics of fullerite, thus creating, in part, a nonmonotonic dependence of the parameter and lattice matrix volume on the impurity saturation temperature. The concentrations of solid solutions C60(CO)x poly- and single crystal samples are determined in the physisorption range for two modes (150 and 250 °C). It is found that the CO impurity has a linear effect on the lattice parameter and the temperature of the orientational transition of fullerite C60.

  15. 16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria.

    PubMed

    Watts, George S; Youens-Clark, Ken; Slepian, Marvin J; Wolk, Donna M; Oshiro, Marc M; Metzger, Gregory S; Dhingra, Dalia; Cranmer, Lee D; Hurwitz, Bonnie L

    2017-09-20

    Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer. Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1-V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0. 1% of the mixture. Sequencing the V1-V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0.1% of a mixture, suggests it has sufficient sensitivity for samples in which important bacteria may be rare. We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. Responses Of Alpine Vegetation To Global Warming: Insights From Comparing Alpine-Restricted And Broad-Ranging Herbs Along Snowmelt Gradients

    NASA Astrophysics Data System (ADS)

    Butz, R. J.; Reinhardt, K. S.; Germino, M. J.; Kueppers, L. M.

    2009-12-01

    Many alpine plant species face habitat fragmentation and loss, and even extinction because their narrow elevation, precipitation, and temperature tolerances limit their geographic distribution. In order to assess the impacts of climate change on sensitive native alpine communities we used a variety of methods to look at the seasonal timing of life stages (phenology) and the stress responses (physiology) of alpine species along a natural environmental gradient at Niwot Ridge in the Colorado Rocky Mountains to address the following question: Will alpine plants be impaired in their existing range as a result of climate change? We collected data on date of snowmelt and vegetative and flowering phenology of all alpine species present from snowmelt to senescence in 80 1m2 plots above treeline. In addition, we measured soil temperature and moisture, plant water potential and leaf-level gas exchange early, mid, and late-season on three alpine-restricted and three broader-ranging alpine species: Geum rossii, Artemisia scopulorum, Carex rupestris, Lewisia pygmaea, Tetraneuris grandiflora, and Sibbaldia procumbens. In 2009, the natural variation in snowmelt timing was 40 days (approximately 5.5 weeks) over the 80 plots. Our results suggest that with earlier snowmelt, the number of vascular species per plot increases. However, this increase is almost exclusively attributable to wider ranging species not restricted to the alpine. Plots with intermediate natural snowmelt dates had a higher diversity of alpine-restricted species, photosynthesis, and water-use efficiency, thereby potentially increasing long-term survival rates amongst alpine species. Water stress increased in all species as the season progressed, especially in plots where snow melted earliest. Photosynthetic productivity and diversity of alpine-restricted species was greatest in plots having intermediate melt dates. These findings suggest that shifts in snowmelt date under a warming climate will likely impact the

  17. Detection of the new cosmopolitan genus Thermoleptolyngbya (Cyanobacteria, Leptolyngbyaceae) using the 16S rRNA gene and 16S-23S ITS region.

    PubMed

    Sciuto, Katia; Moro, Isabella

    2016-12-01

    Cyanobacteria are widespread prokaryotes that are able to live in extreme conditions such as thermal springs. Strains attributable to the genus Leptolyngbya are among the most common cyanobacteria sampled from thermal environments. Leptolyngbya is a character-poor taxon that was demonstrated to be polyphyletic based on molecular analyses. The recent joining of 16S rRNA gene phylogenies with 16S-23S ITS secondary structure analysis is a useful approach to detect new cryptic taxa and has led to the separation of new genera from Leptolyngbya and to the description of new species inside this genus and in other related groups. In this study, phylogenetic investigations based on both the 16S rRNA gene and the 16S-23S ITS region were performed alongside 16S rRNA and 16S-23S ITS secondary structure analyses on cyanobacteria of the family Leptolyngbyaceae. These analyses focused on filamentous strains sampled from thermal springs with a morphology ascribable to the genus Leptolyngbya. The phylogenetic reconstructions showed that the Leptolyngbya-like thermal strains grouped into a monophyletic lineage that was distinct from Leptolyngbya. The 16S-23S ITS secondary structure results supported the separation of this cluster. A new genus named Thermoleptolyngbya was erected to encompass these strains, and two species were described inside this new taxon: T. albertanoae and T. oregonensis. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. The DBA.2 Mouse Is Susceptible to Disease following Infection with a Broad, but Limited, Range of Influenza A and B Viruses▿

    PubMed Central

    Pica, Natalie; Iyer, Arun; Ramos, Irene; Bouvier, Nicole M.; Fernandez-Sesma, Ana; García-Sastre, Adolfo; Lowen, Anice C.; Palese, Peter; Steel, John

    2011-01-01

    We assessed the relative susceptibilities to disease of the DBA.2 and C57BL/6 mouse models upon infection with a range of influenza A and B viruses. DBA.2 mice were more susceptible to disease upon inoculation with human H1N1 influenza A virus strains, several swine influenza viruses, and influenza B viruses but were not overtly susceptible to infection with human seasonal H3N2 strains. Hemagglutination inhibition and immunoglobulin isotype profiling indicated that DBA.2 and C57BL/6 mice generate comparable humoral responses upon equivalent 50% mouse lethal dose (MLD50) challenges with influenza virus. Our data demonstrate the utility of DBA.2 mice for the elucidation of influenza virus pathogenicity determinants and the testing of influenza vaccines. PMID:21917963

  19. A measurement of the energy spectra and relative abundance of the cosmic-ray H and He isotopes over a broad energy range

    NASA Technical Reports Server (NTRS)

    Webber, W. R.; Yushak, S. M.

    1983-01-01

    The measurements reported of these isotopes were made using two sets of detectors during the same minimum modulation period in 1977. One measurement was made with a balloon-borne telescope, the other with telescopes on the Voyager spacecraft. It is noted that together they provide the widest energy range yet available for studying these isotopes: 14-150 MeV per nucleon for H2 and 10-290 MeV per nucleon for He-3. The simultaneous helium isotope observations are used to give a mutually consistent picture of galactic propagation and solar modulation. The data define the form of the interstellar H-1 and He-4 spectra, an interstellar matter path length for both H-1 and He-4, and a total residual modulation for He-4. The H-2 observations suggest a picture that is very similar for the galactic propagation of H-1 and He-4.

  20. 16S-23S rDNA internal transcribed spacer regions in four Proteus species.

    PubMed

    Cao, Boyang; Wang, Min; Liu, Lei; Zhou, Zhemin; Wen, Shaoping; Rozalski, Antoni; Wang, Lei

    2009-04-01

    Proteus is a Gram-negative, facultative anaerobic bacterium. In this study, 813 Proteus 16S-23S rDNA internal transcribed spacer (ITS) sequences were determined from 46 Proteus strains, including 388 ITS from 22 P. mirabilis strains, 211 ITS from 12 P. vulgaris strains, 169 ITS from 10 P. penneri strains, and 45 ITS from 2 P. myxofaciens strains. The Proteus strains carry mainly two types of ITS, ITS(Glu) (containing tRNA(Glu (UUC)) gene) and ITS(Ile+Ala) (containing tRNA(Ile (GAU)) and tRNA(Ala (UGC)) gene), and are in the forms of 28 variants with 25 genomic origins. The ITS sequences are a mosaic-like structure consisting of three conservative regions and two variable regions. The nucleotide identity of ITS subtypes in strains of the same species ranges from 96.2% to 100%. The divergence of Proteus ITS divergence was most likely due to intraspecies recombinations or horizontal transfers of sequence blocks. The phylogenetic relationship deduced from the second variable region of ITS sequences of the three facultative human pathogenic species P. mirabilis, P. vulgaris and P. penneri is similar with that based on 16S rDNA sequences, but has higher resolution to differentiate closely related P. vulgaris and P. penneri. This study is the first comprehensive study of ITS in four Proteus species and laid solid foundation for the development of high-throughput technology for quick and accurate identification of the important foodborne and nosocomial pathogens.

  1. Emergence and Dissemination of Enterobacteriaceae Isolates Producing CTX-M-1-Like Enzymes in Spain Are Associated with IncFII (CTX-M-15) and Broad-Host-Range (CTX-M-1, -3, and -32) Plasmids▿

    PubMed Central

    Novais, Ângela; Cantón, Rafael; Moreira, Raquel; Peixe, Luísa; Baquero, Fernando; Coque, Teresa M.

    2007-01-01

    The spread of CTX-M-1-like enzymes in Spain is associated with particular plasmids of broad-host-range IncN (blaCTX-M-32, blaCTX-M-1), IncL/M (blaCTX-M-1), and IncA/C2 (blaCTX-M-3) or narrow-host-range IncFII (blaCTX-M-15). The identical genetic surroundings of blaCTX-M-32 and blaCTX-M-1 and their locations on related 40-kb IncN plasmids indicate the in vivo evolution of this element. PMID:17145793

  2. Comparison of bacterial culture and 16S rRNA community profiling by clonal analysis and pyrosequencing for the characterization of the dentine caries-associated microbiome

    PubMed Central

    Schulze-Schweifing, Kathrin; Banerjee, Avijit; Wade, William G.

    2014-01-01

    Culture-independent analyses have greatly expanded knowledge regarding the composition of complex bacterial communities including those associated with oral diseases. A consistent finding from such studies, however, has been the under-reporting of members of the phylum Actinobacteria. In this study, five pairs of broad range primers targeting 16S rRNA genes were used in clonal analysis of 6 samples collected from tooth lesions involving dentine in subjects with active caries. Samples were also subjected to cultural analysis and pyrosequencing by means of the 454 platform. A diverse bacterial community of 229 species-level taxa was revealed by culture and clonal analysis, dominated by representatives of the genera Prevotella, Lactobacillus, Selenomonas, and Streptococcus. The five most abundant species were: Lactobacillus gasseri, Prevotella denticola, Alloprevotella tannerae, S. mutans and Streptococcus sp. HOT 070, which together made up 31.6 % of the sequences. Two samples were dominated by lactobacilli, while the remaining samples had low numbers of lactobacilli but significantly higher numbers of Prevotella species. The different primer pairs produced broadly similar data but proportions of the phylum Bacteroidetes were significantly higher when primer 1387R was used. All of the primer sets underestimated the proportion of Actinobacteria compared to culture. Pyrosequencing analysis of the samples was performed to a depth of sequencing of 4293 sequences per sample which were identified to 264 species-level taxa, and resulted in significantly higher coverage estimates than the clonal analysis. Pyrosequencing, however, also underestimated the relative abundance of Actinobacteria compared to culture. PMID:25429361

  3. Using a Pareto-optimal solution set to characterize trade-offs between a broad range of values and preferences in climate risk management

    NASA Astrophysics Data System (ADS)

    Garner, Gregory; Reed, Patrick; Keller, Klaus

    2015-04-01

    Integrated assessment models (IAMs) are often used to inform the design of climate risk management strategies. Previous IAM studies have broken important new ground on analyzing the effects of parametric uncertainties, but they are often silent on the implications of uncertainties regarding the problem formulation. Here we use the Dynamic Integrated model of Climate and the Economy (DICE) to analyze the effects of uncertainty surrounding the definition of the objective(s). The standard DICE model adopts a single objective to maximize a weighted sum of utilities of per-capita consumption. Decision makers, however, are often concerned with a broader range of values and preferences that may be poorly captured by this a priori definition of utility. We reformulate the problem by introducing three additional objectives that represent values such as (i) reliably limiting global average warming to two degrees Celsius and minimizing (ii) the costs of abatement and (iii) the climate change damages. We use advanced multi-objective optimization methods to derive a set of Pareto-optimal solutions over which decision makers can trade-off and assess performance criteria a posteriori. We illustrate the potential for myopia in the traditional problem formulation and discuss the capability of this multiobjective formulation to provide decision support.

  4. Transcription reporters that shuttle cloned DNA between high-copy Escherichia coli plasmids and low-copy broad-host-range plasmids.

    PubMed

    Ouimet, M C; Marczynski, G T

    2000-09-01

    We describe and apply lacZ transcription reporter plasmids designed for both biochemical analyses requiring high DNA yield and physiological studies requiring low gene dosage. Standard DNA ligations are performed at seven unique restriction sites 5' to the lacZ gene on high-copy ColE1 plasmids suitable for double- or single-strand DNA sequencing. A divergent gusA transcription reporter is included and serves as an internal control. Rec(+) Escherichia coli cells readily shuttle DNA placed between gusA and lacZ by allelic exchange with pRK290-based plasmids that subsequently conjugate and replicate in most gram-negative bacteria. We applied this system to study Caulobacter crescentus cell cycle promoters directed by the CtrA response-regulator protein. Synthetic oligonucleotides were ligated to create altered CtrA binding sites and corresponding promoters with varied transcription strength. We also document the phenomenon of long-range promoter interference. A strong promoter can repress up to twofold the transcription from a divergent promoter located 100 bp away. However, the cell cycle timing of both promoters is not changed. Additional applications of our system and theoretical aspects of promoter organization are discussed. Copyright 2000 Academic Press.

  5. Prevalent presence of periodic actin-spectrin-based membrane skeleton in a broad range of neuronal cell types and animal species.

    PubMed

    He, Jiang; Zhou, Ruobo; Wu, Zhuhao; Carrasco, Monica A; Kurshan, Peri T; Farley, Jonathan E; Simon, David J; Wang, Guiping; Han, Boran; Hao, Junjie; Heller, Evan; Freeman, Marc R; Shen, Kang; Maniatis, Tom; Tessier-Lavigne, Marc; Zhuang, Xiaowei

    2016-05-24

    Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal- and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens.

  6. Short-term changes in bone formation markers following growth hormone (GH) treatment in short prepubertal children with a broad range of GH secretion.

    PubMed

    Andersson, Björn; Swolin-Eide, Diana; Magnusson, Per; Albertsson-Wikland, Kerstin

    2015-01-01

    Growth hormone (GH) promotes longitudinal growth and bone modelling/remodelling. This study investigated the relationship between levels of bone formation markers and growth during GH treatment in prepubertal children with widely ranging GH secretion levels. The study group comprised 113 short prepubertal children (mean age ± SD, 9·37 ± 2·13 years; 99 boys) on GH treatment (33·0 ± 0·06 μg/kg/day) for 1 year. Blood samples were taken at baseline and 1 and 2 weeks, 1 and 3 months, and 1 year after treatment start. Intact amino-terminal propeptide of type I procollagen (PINP), bone-specific alkaline phosphatase (BALP) and osteocalcin were measured using an automated IDS-iSYS immunoassay system. Intact amino-terminal propeptide of type I procollagen (PINP), BALP and osteocalcin, increased in the short-term during GH treatment. PINP after 1 week (P = 0·00077), and BALP and osteocalcin after 1 month (P < 0·0001 and P = 0·0043, respectively). PINP levels at 1 and 3 months correlated positively, and osteocalcin levels at 1 week and percentage change after 1 month correlated negatively, with first year growth response. No significant correlations were found between BALP and first year growth. Multiple regression analysis showed that bone marker levels together with auxological data and insulin-like growth factor binding protein-3 explained the variation in first year growth response to 36% at start, 32% after 2 weeks and 48% at 3 months. Short-term increases in levels of the bone formation markers PINP, BALP and osteocalcin showed different temporal patterns, but all correlated with first year growth response during GH treatment. These markers may be a useful addition to existing prediction models for growth response. © 2014 John Wiley & Sons Ltd.

  7. Prevalent presence of periodic actin–spectrin-based membrane skeleton in a broad range of neuronal cell types and animal species

    PubMed Central

    He, Jiang; Zhou, Ruobo; Wu, Zhuhao; Carrasco, Monica A.; Kurshan, Peri T.; Farley, Jonathan E.; Simon, David J.; Wang, Guiping; Han, Boran; Hao, Junjie; Heller, Evan; Freeman, Marc R.; Shen, Kang; Maniatis, Tom; Tessier-Lavigne, Marc

    2016-01-01

    Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal- and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens. PMID:27162329

  8. Childhood ADHD is Strongly Associated with a Broad Range of Psychiatric Disorders during Adolescence: a Population-Based Birth Cohort Study

    PubMed Central

    Yoshimasu, Kouichi; Barbaresi, William J.; Colligan, Robert C.; Voigt, Robert G.; Killian, Jill M.; Weaver, Amy L.; Katusic, Slavica K.

    2013-01-01

    Background To evaluate associations between ADHD and comorbid psychiatric disorders using research-identified incident cases of ADHD and population-based controls. Methods Subjects included a birth cohort of all children born 1976–1982 remaining in Rochester, MN after age five (n = 5718). Among them we identified 379 ADHD incident cases and 758 age-sex matched non-ADHD controls, passively followed to age 19 years. All psychiatric diagnoses were identified and abstracted, but only those confirmed by qualified medical professionals were included in the analysis. For each psychiatric disorder, cumulative incidence rates for subjects with and without ADHD were estimated using the Kaplan-Meier method. Corresponding hazard ratios (HR) were estimated using Cox models adjusted for gender and mother’s age and education at the subject’s birth. The association between ADHD and the likelihood of having an internalizing or externalizing disorder was summarized by estimating odds ratios (OR). Results ADHD was associated with a significantly increased risk of adjustment disorders (HR=3.88), conduct/oppositional defiant disorder (HR=9.54), mood disorders (HR=3.67), anxiety disorders (HR=2.94), tic disorders (HR=6.53), eating disorders (HR=5.68), personality disorders (HR=5.80), and substance-related disorders (HR=4.03). When psychiatric comorbidities were classified on the internalization-externalization dimension, ADHD was strongly associated with coexisting internalizing/externalizing (OR=10.6), or externalizing-only (OR=10.0) disorders. Conclusion This population-based study confirms that children with ADHD are at significantly increased risk for a wide range of psychiatric disorders. Besides treating the ADHD, clinicians should identify and provide appropriate treatment for psychiatric comorbidities. PMID:22647074

  9. Segmentation of brain magnetic resonance images based on multi-atlas likelihood fusion: testing using data with a broad range of anatomical and photometric profiles

    PubMed Central

    Tang, Xiaoying; Crocetti, Deana; Kutten, Kwame; Ceritoglu, Can; Albert, Marilyn S.; Mori, Susumu; Mostofsky, Stewart H.; Miller, Michael I.

    2015-01-01

    We propose a hierarchical pipeline for skull-stripping and segmentation of anatomical structures of interest from T1-weighted images of the human brain. The pipeline is constructed based on a two-level Bayesian parameter estimation algorithm called multi-atlas likelihood fusion (MALF). In MALF, estimation of the parameter of interest is performed via maximum a posteriori estimation using the expectation-maximization (EM) algorithm. The likelihoods of multiple atlases are fused in the E-step while the optimal estimator, a single maximizer of the fused likelihoods, is then obtained in the M-step. There are two stages in the proposed pipeline; first the input T1-weighted image is automatically skull-stripped via a fast MALF, then internal brain structures of interest are automatically extracted using a regular MALF. We assess the performance of each of the two modules in the pipeline based on two sets of images with markedly different anatomical and photometric contrasts; 3T MPRAGE scans of pediatric subjects with developmental disorders vs. 1.5T SPGR scans of elderly subjects with dementia. Evaluation is performed quantitatively using the Dice overlap as well as qualitatively via visual inspections. As a result, we demonstrate subject-level differences in the performance of the proposed pipeline, which may be accounted for by age, diagnosis, or the imaging parameters (particularly the field strength). For the subcortical and ventricular structures of the two datasets, the hierarchical pipeline is capable of producing automated segmentations with Dice overlaps ranging from 0.8 to 0.964 when compared with the gold standard. Comparisons with other representative segmentation algorithms are presented, relative to which the proposed hierarchical pipeline demonstrates comparative or superior accuracy. PMID:25784852

  10. Coupling and optimisation of online nuclear magnetic resonance spectroscopy and mass spectrometry for process monitoring to cover the broad range of process concentration.

    PubMed

    Blanazs, Alexander; Bristow, Tony W T; Coombes, Steven R; Corry, Tom; Nunn, Mike; Ray, Andrew D

    2017-04-01

    Real time online monitoring of chemical processes can be carried out by a number of analytical techniques, including optical and vibrational spectroscopies, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). As each technique has unique advantages and challenges, combinations are an attractive option. The combination of a 500-MHz (1) H NMR and a small footprint mass spectrometer to monitor a batch reaction at process concentration was investigated. The mass spectrometer was coupled into the flow path of an online reaction monitoring NMR. Reaction mixture was pumped from a 100-ml vessel to an NMR flow tube before returning to the vessel. Small aliquots were diverted into a sampling make-up flow using an active flow splitter and passed to the mass spectrometer. Advantages of the combination were observed. (1) H NMR was ideal for quantitation of high level components, whereas MS showed a greater capability for detecting those at low level. In preliminary experiments MS produced a limited linear relationship with concentration (0.02% to 2% relative concentration, 0.01 mg/ml-1.25 mg/ml), because of signal saturation at the higher concentrations. NMR was unable to detect components below 0.1% relative to concentration maximum. Optimisation of sample transfer to the MS extended the linearity to 10% relative to the concentration maximum. Therefore, the combination of online NMR and MS allows both qualitative and quantitative analysis of reaction components over the full process range. The application of the combination was demonstrated by monitoring a batch chemical reaction and this is described. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Complete Genome Sequence of the N2-Fixing Broad Host Range Endophyte Klebsiella pneumoniae 342 and Virulence Predictions Verified in Mice

    PubMed Central

    Fouts, Derrick E.; Tyler, Heather L.; DeBoy, Robert T.; Daugherty, Sean; Ren, Qinghu; Badger, Jonathan H.; Durkin, Anthony S.; Huot, Heather; Shrivastava, Susmita; Kothari, Sagar; Dodson, Robert J.; Mohamoud, Yasmin; Khouri, Hoda; Roesch, Luiz F. W.; Krogfelt, Karen A.; Struve, Carsten; Triplett, Eric W.; Methé, Barbara A.

    2008-01-01

    We report here the sequencing and analysis of the genome of the nitrogen-fixing endophyte, Klebsiella pneumoniae 342. Although K. pneumoniae 342 is a member of the enteric bacteria, it serves as a model for studies of endophytic, plant-bacterial associations due to its efficient colonization of plant tissues (including maize and wheat, two of the most important crops in the world), while maintaining a mutualistic relationship that encompasses supplying organic nitrogen to the host plant. Genomic analysis examined K. pneumoniae 342 for the presence of previously identified genes from other bacteria involved in colonization of, or growth in, plants. From this set, approximately one-third were identified in K. pneumoniae 342, suggesting additional factors most likely contribute to its endophytic lifestyle. Comparative genome analyses were used to provide new insights into this question. Results included the identification of metabolic pathways and other features devoted to processing plant-derived cellulosic and aromatic compounds, and a robust complement of transport genes (15.4%), one of the highest percentages in bacterial genomes sequenced. Although virulence and antibiotic resistance genes were predicted, experiments conducted using mouse models showed pathogenicity to be attenuated in this strain. Comparative genomic analyses with the presumed human pathogen K. pneumoniae MGH78578 revealed that MGH78578 apparently cannot fix nitrogen, and the distribution of genes essential to surface attachment, secretion, transport, and regulation and signaling varied between each genome, which may indicate critical divergences between the strains that influence their preferred host ranges and lifestyles (endophytic plant associations for K. pneumoniae 342 and presumably human pathogenesis for MGH78578). Little genome information is available concerning endophytic bacteria. The K. pneumoniae 342 genome will drive new research into this less-understood, but important category

  12. Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible Expression Vector with Tight Repression and Broad Dynamic Range.

    PubMed

    Weaver, Keith E; Chen, Yuqing; Miiller, Elly M; Johnson, Jake N; Dangler, Alex A; Manias, Dawn A; Clem, Aaron M; Schjodt, Daniel J; Dunny, Gary M

    2017-06-15

    Tools for regulated gene expression in Enterococcus faecalis are extremely limited. In this report, we describe the construction of an expression vector for E. faecalis, designated pCIE, utilizing the PQ pheromone-responsive promoter of plasmid pCF10. We demonstrate that this promoter is tightly repressed, responds to nanogram quantities of the peptide pheromone, and has a large dynamic range. To demonstrate its utility, the promoter was used to control expression of the toxic peptides of two par family toxin-antitoxin (TA) loci present in E. faecalis, parpAD1 of the pAD1 plasmid and parEF0409 located on the E. faecalis chromosome. The results demonstrated differences in the modes of regulation of toxin expression and in the effects of toxins of these two related systems. We anticipate that this vector will be useful for further investigation of par TA system function as well as the regulated expression of other genes in E. faecalisIMPORTANCEE. faecalis is an important nosocomial pathogen and a model organism for examination of the genetics and physiology of Gram-positive cocci. While numerous genetic tools have been generated for the manipulation of this organism, vectors for the regulated expression of cloned genes remain limited by high background expression and the use of inducers with undesirable effects on the cell. Here we demonstrate that the PQ pheromone-responsive promoter is repressed tightly enough to allow cloning of TA system toxins and evaluate their effects at very low induction levels. This tool will allow us to more fully examine TA system function in E. faecalis and to further elucidate its potential roles in cell physiology. Copyright © 2017 American Society for Microbiology.

  13. Superheating and melting within aluminum core-oxide shell nanoparticles for a broad range of heating rates: multiphysics phase field modeling.

    PubMed

    Hwang, Yong Seok; Levitas, Valery I

    2016-10-19

    The external surface of metallic particles is usually covered by a thin and strong oxide shell, which significantly affects superheating and melting of particles. The effects of geometric parameters and heating rate on characteristic melting and superheating temperatures and melting behavior of aluminum nanoparticles covered by an oxide shell were studied numerically. For this purpose, the multiphysics model that includes the phase field model for surface melting, a dynamic equation of motion, a mechanical model for stress and strain simulations, interface and surface stresses, and the thermal conduction model including thermoelastic and thermo-phase transformation coupling as well as transformation dissipation rate was formulated. Several nontrivial phenomena were revealed. In comparison with a bare particle, the pressure generated in a core due to different thermal expansions of the core and shell and transformation volumetric expansion during melting, increases melting temperatures with the Clausius-Clapeyron factor of 60 K GPa(-1). For the heating rates Q ≤ 10(9) K s(-1), melting temperatures (surface and bulk start and finish melting temperatures, and maximum superheating temperature) are independent of Q. For Q ≥ 10(12) K s(-1), increasing Q generally increases melting temperatures and temperature for the shell fracture. Unconventional effects start for Q ≥ 10(12) K s(-1) due to kinetic superheating combined with heterogeneous melting and geometry. The obtained results are applied to shed light on the initial stage of the melt-dispersion-mechanism of the reaction of Al nanoparticles. Various physical phenomena that promote or suppress melting and affect melting temperatures and temperature of the shell fracture for different heating-rate ranges are summarized in the corresponding schemes.

  14. Global effects of the DEAD-box RNA helicase DeaD (CsdA) on gene expression over a broad range of temperatures

    PubMed Central

    Vakulskas, Christopher A.; Pannuri, Archana; Cortés-Selva, Diana; Zere, Tesfalem R.; Ahmer, Brian M.; Babitzke, Paul; Romeo, Tony

    2014-01-01

    Summary In Escherichia coli, activity of the global regulatory RNA binding protein CsrA is antagonized by two noncoding sRNAs, CsrB and CsrC, which sequester it away from its lower affinity mRNA targets. Transcription of csrB/C requires the BarA-UvrY two component signal transduction system, which responds to short chain carboxylates. We show that two DEAD-box RNA helicases, DeaD and SrmB, activate csrB/C expression by different pathways. DeaD facilitates uvrY translation by counteracting the inhibitory effect of long distance basepairing between the uvrY mRNA leader and coding region, while SrmB does not affect UvrY or UvrY-phosphate levels. Contrary to the prevailing notion that these helicases act primarily at low temperatures, DeaD and SrmB activated csrB expression over a wide temperature range. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) revealed in vivo interactions of DeaD with 39 mRNAs, including those of uvrY and 9 other regulatory genes. Studies on the expression of several of the identified genes revealed regulatory effects of DeaD in all cases and diverse temperature response patterns. Our findings uncover an expanded regulatory role for DeaD, which is mediated through novel mRNA targets, important global regulators and under physiological conditions that were considered to be incompatible with its function. PMID:24708042

  15. Purification, biochemical characterization and antifungal activity of a novel Aspergillus tubingensis glucose oxidase steady on broad range of pH and temperatures.

    PubMed

    Kriaa, Mouna; Hammami, Inès; Sahnoun, Mouna; Azebou, Manel Cheffi; Triki, Mohamed Ali; Kammoun, Radhouane

    2015-11-01

    This study was carried out to evaluate the in vitro and in vivo antifungal efficiency of Aspergillus tubingensis CTM 507 glucose oxidase (GOD) against plant pathogenic fungi. GOD displayed a wide inhibitory spectrum toward different fungi at a concentration of 20 AU. The GOD had a strong inhibitor effect on mycelia growth and spore germination of Pythium ultimum. Interestingly, the GOD exhibited a potent in vivo antifungal effect against P. ultimum responsible for potato plants disease. The antifungal GOD was purified 13-fold with 27 % yield and a specific activity of 3435 U/mg. The relative molecular mass of the GOD was 180 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The GOD activity was optimum at pH 4.5 and 60 °C. It was found to be stable over a large pH range (3-9). It also displayed a marked thermostability with a 50-min half-life at 65 °C. The 10 residues of the N-terminal sequence of the purified GOD (S-K-G-S-A-V-T-T-P-D) showed no homology to the other reported GOD, identifying a novel GOD. FTIR spectroscopic analysis revealed the presence of C-O and C=O groups corresponding to a D-glucono-lactone. The findings indicated that GOD is the first A. tubingensis-produced fungicide ever reported to exhibit such promising biological properties. It could become a natural alternative to synthetic fungicides to control certain important plant microbial diseases.

  16. Identification in gelada baboons (Theropithecus gelada) of a distinct simian T-cell lymphotropic virus type 3 with a broad range of Western blot reactivity.

    PubMed

    Van Dooren, Sonia; Shanmugam, Vedapuri; Bhullar, Vinod; Parekh, Bharat; Vandamme, Anne-Mieke; Heneine, Walid; Switzer, William M

    2004-02-01

    STLV-3-infected primates show a range of HTLV-like WB profiles and have an ancient origin, further studies using STLV-3-specific testing are required to determine whether STLV-3 infects humans, especially in regions of Africa where STLV-3 is endemic.

  17. Internal management of non-structural carbohydrate resources in apple leaves and branch wood under a broad range of sink and source manipulations.

    PubMed

    Naschitz, Shaul; Naor, Amos; Genish, Shani; Wolf, Shmuel; Goldschmidt, Eliezer E

    2010-06-01

    Apple (Malus x domestica Borkh.) grown in a Mediterranean climate depends on regular irrigation throughout the growing season. The objective of the current study was to elucidate the changes in carbohydrate storage and utilization by mature, field-grown apple trees in response to water availability to the trees and to the level of cropping. Fourteen-year-old apple trees cv. 'Golden Delicious' were grown under various combinations of irrigation rate (11, 33 or 77 l day(-)(1) per tree) and crop level ( approximately 100, approximately 300 or >1000 fruits per tree) beginning 47 days after full bloom (DAFB). Non-structural carbohydrate concentrations were measured at 78 (leaves and branch wood), 102 (leaves), 183 (branch wood) and 214 (branch wood) DAFB. Midday stem water potential (SWP) was measured at 2-week intervals between June and October. Trunk cross-sectional area was measured 47 and 265 DAFB. At harvest, 139 DAFB, the fruits of each tree were counted and weighed. SWP at 102 DAFB ranged between -0.6 and -2.7 MPa. Fruit fresh weight at harvest was positively related to SWP measured 37 days before harvest with distinct slopes for light/intermediate and heavy crop levels. Leaf and branch wood starch concentrations 78 and 102 DAFB were positively related to irrigation rate and negatively related to crop level. Mean fruit weight at harvest was positively related to branch wood starch concentration and neared maximum at a concentration of 40 mg g(-)(1) dry weight. Branch wood starch concentration recovered after harvest, especially in water-stressed trees. Sorbitol concentration was negatively related to irrigation rate. The sorbitol-to-starch concentration ratio in leaves at 102 DAFB was closely proportional to SWP. It is suggested that branch wood starch concentration represents the overall balance between carbon sources and sinks and may therefore serve as a reliable indicator of photo-assimilate availability. In water-stressed trees, sorbitol is prioritized over

  18. Complete ecological isolation and cryptic diversity in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences

    PubMed Central

    Hahn, Martin W; Jezberová, Jitka; Koll, Ulrike; Saueressig-Beck, Tanja; Schmidt, Johanna

    2016-01-01

    Transplantation experiments and genome comparisons were used to determine if lineages of planktonic Polynucleobacter almost indistinguishable by their 16S ribosomal RNA (rRNA) sequences differ distinctively in their ecophysiological and genomic traits. The results of three transplantation experiments differing in complexity of biotic interactions revealed complete ecological isolation between some of the lineages. This pattern fits well to the previously detected environmental distribution of lineages along chemical gradients, as well as to differences in gene content putatively providing adaptation to chemically distinct habitats. Patterns of distribution of iron transporter genes across 209 Polynucleobacter strains obtained from freshwater systems and representing a broad pH spectrum further emphasize differences in habitat-specific adaptations. Genome comparisons of six strains sharing ⩾99% 16S rRNA similarities suggested that each strain represents a distinct species. Comparison of sequence diversity among genomes with sequence diversity among 240 cultivated Polynucleobacter strains indicated a large cryptic species complex not resolvable by 16S rRNA sequences. The revealed ecological isolation and cryptic diversity in Polynucleobacter bacteria is crucial in the interpretation of diversity studies on freshwater bacterioplankton based on ribosomal sequences. PMID:26943621

  19. Thermococcus piezophilus sp. nov., a novel hyperthermophilic and piezophilic archaeon with a broad pressure range for growth, isolated from a deepest hydrothermal vent at the Mid-Cayman Rise.

    PubMed

    Dalmasso, Cécile; Oger, Philippe; Selva, Gwendoline; Courtine, Damien; L'Haridon, Stéphane; Garlaschelli, Alexandre; Roussel, Erwan; Miyazaki, Junichi; Reveillaud, Julie; Jebbar, Mohamed; Takai, Ken; Maignien, Lois; Alain, Karine

    2016-10-01

    A novel strictly anaerobic, hyperthermophilic archaeon, designated strain CDGS(T), was isolated from a deep-sea hydrothermal vent in the Cayman Trough at 4964m water depth. The novel isolate is obligate anaerobe and grows chemoorganoheterotrophically with stimulation of growth by sulphur containing compounds. Its growth is optimal at 75°C, pH 6.0 and under a pressure of 50MPa. It possesses the broadest hydrostatic pressure range for growth that has ever been described for a microorganism. Its genomic DNA G+C content is 51.11mol%. The novel isolate belongs to the genus Thermococcus. Phylogenetic analyses indicated that it is most closely related to Thermococcus barossii DSM17882(T) based on its 16S rRNA gene sequence, and to 'Thermococcus onnurineus' NA1 based on its whole genome sequence. The average nucleotide identity scores with these strains are 77.66% for T. barossii and 84.84% for 'T. onnurineus', respectively. Based on the draft whole genome sequence and phenotypic characteristics, strain CDGS(T) is suggested to be separated into a novel species within the genus Thermococcus, with proposed name Thermococcus piezophilus (type strain CDGS(T)=ATCC TSD-33(T)=UBOCC 3296(T)). Copyright © 2016 Elsevier GmbH. All rights reserved.

  20. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

    SciTech Connect

    Walters, William; Hyde, Embriette R.; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A.; Jansson, Janet K.; Caporaso, J. Gregory; Fuhrman, Jed A.; Apprill, Amy; Knight, Rob; Bik, Holly

    2015-12-22

    ABSTRACT

    Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection ofThaumarchaeotaand clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies.

    ImportanceWe continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to

  1. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

    PubMed Central

    Walters, William; Hyde, Embriette R.; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A.; Jansson, Janet K.; Caporaso, J. Gregory; Fuhrman, Jed A.; Apprill, Amy

    2015-01-01

    ABSTRACT Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers

  2. Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses

    PubMed Central

    Chen, Jiazhen; Miao, Xinyu; Xu, Meng; He, Junlin; Xie, Yi; Wu, Xingwen; Chen, Gang; Yu, Liying; Zhang, Wenhong

    2015-01-01

    Background Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported. Methods The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains. Results Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed. Conclusion Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3–100%. However, the

  3. Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses.

    PubMed

    Chen, Jiazhen; Miao, Xinyu; Xu, Meng; He, Junlin; Xie, Yi; Wu, Xingwen; Chen, Gang; Yu, Liying; Zhang, Wenhong

    2015-01-01

    Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported. The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains. Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed. Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3-100%. However, the inter-species similarities were

  4. Structure of E. coli 16S RNA elucidated by psoralen crosslinking

    SciTech Connect

    Thompson, J.F.; Hearst, J.E.

    1983-04-01

    E. coli 16S RNA in solution was photoreacted with hydroxymethyltrimethylpsoralen and long wave ultraviolet light. Positions of crosslinks were determined to high resolution by partially digesting the RNA with T/sub 1/ RNase, separating the crosslinked fragments by two-dimensional gel electrophoresis, reversing the crosslink, and sequencing the separated fragments. This method yielded the locations of crosslinks to +/-15 nucleotides. Even finer placement has been made on the basis of our knowledge of psoralen reactivity. Thirteen unique crosslinks were mapped. Seven crosslinks confirmed regions of secondary structure which had been predicted in published phylogenetic models, three crosslinks discriminated between phylogenetic models, and three proved the existence of new structures. The new structures were all long-range interactions which appear to be in dynamic equilibrium with local secondary structure. Because this technique yields direct information about the secondary structure of large RNAs, it should prove invaluable in studying the structure of other RNAs of all sizes.

  5. Broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 and specific detection of Akabane, Aino and Peaton viruses by newly developed multiple TaqMan assays.

    PubMed

    Shirafuji, Hiroaki; Yazaki, Ryu; Shuto, Yozo; Yanase, Tohru; Kato, Tomoko; Ishikura, Youji; Sakaguchi, Zenjiro; Suzuki, Moemi; Yamakawa, Makoto

    2015-12-01

    TaqMan assays were developed for the broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 in the genus Orthobunyavirus and also for the specific detection of three viruses in the lineage, Akabane, Aino and Peaton viruses (AKAV, AINOV and PEAV, respectively). A primer and probe set was designed for the broad-range detection of Simbu serogroup lineage 1 (Pan-Simbu1 set) mainly targeting AKAV, AINOV, PEAV, Sathuperi and Shamonda viruses (SATV and SHAV), and the forward and reverse primers of the Pan-Simbu1 set were also used for the specific detection of AKAV with another probe (AKAV-specific set). In addition, two more primer and probe sets were designed for AINOV- and PEAV-specific detection, respectively (AINOV- and PEAV-specific sets). All of the four primer and probe sets successfully detected targeted viruses, and thus broad-range and specific detection of all the targeted viruses can be achieved by using two multiplex assays and a single assay in a dual (two-color) assay format when another primer and probe set for a bovine β-actin control is also used. The assays had an analytical sensitivity of 10 copies/tube for AKAV, at least 100 copies/tube for AINOV, 100 copies/tube for PEAV, one copy/tube for SATV and at least 10 copies/tube for SHAV, respectively. Diagnostic sensitivity of the assays was tested with field-collected bovine samples, and the results suggested that the sensitivity was higher than that of a conventional RT-PCR. These data indicate that the newly developed TaqMan assays will be useful tools for the diagnosis and screening of field-collected samples for infections of AKAV and several other arboviruses belonging to the Simbu serogroup lineage 1.

  6. Centrality evolution of the charged-particle pseudorapidity density over a broad pseudorapidity range in Pb-Pb collisions at √{sNN} = 2.76 TeV

    NASA Astrophysics Data System (ADS)

    Adam, J.; Adamová, D.; Aggarwal, M. M.; Aglieri Rinella, G.; Agnello, M.; Agrawal, N.; Ahammed, Z.; Ahn, S. U.; Aiola, S.; Akindinov, A.; Alam, S. N.; Aleksandrov, D.; Alessandro, B.; Alexandre, D.; Alfaro Molina, R.; Alici, A.; Alkin, A.; Almaraz, J. R. M.; Alme, J.; Alt, T.; Altinpinar, S.; Altsybeev, I.; Alves Garcia Prado, C.; Andrei, C.; Andronic, A.; Anguelov, V.; Anielski, J.; Antičić, T.; Antinori, F.; Antonioli, P.; Aphecetche, L.; Appelshäuser, H.; Arcelli, S.; Arnaldi, R.; Arnold, O. W.; Arsene, I. C.; Arslandok, M.; Audurier, B.; Augustinus, A.; Averbeck, R.; Azmi, M. D.; Badalà, A.; Baek, Y. W.; Bagnasco, S.; Bailhache, R.; Bala, R.; Baldisseri, A.; Baral, R. C.; Barbano, A. M.; Barbera, R.; Barile, F.; Barnaföldi, G. G.; Barnby, L. S.; Barret, V.; Bartalini, P.; Barth, K.; Bartke, J.; Bartsch, E.; Basile, M.; Bastid, N.; Basu, S.; Bathen, B.; Batigne, G.; Batista Camejo, A.; Batyunya, B.; Batzing, P. C.; Bearden, I. G.; Beck, H.; Bedda, C.; Behera, N. K.; Belikov, I.; Bellini, F.; Bello Martinez, H.; Bellwied, R.; Belmont, R.; Belmont-Moreno, E.; Belyaev, V.; Bencedi, G.; Beole, S.; Berceanu, I.; Bercuci, A.; Berdnikov, Y.; Berenyi, D.; Bertens, R. A.; Berzano, D.; Betev, L.; Bhasin, A.; Bhat, I. R.; Bhati, A. K.; Bhattacharjee, B.; Bhom, J.; Bianchi, L.; Bianchi, N.; Bianchin, C.; Bielčík, J.; Bielčíková, J.; Bilandzic, A.; Biswas, R.; Biswas, S.; Bjelogrlic, S.; Blair, J. T.; Blau, D.; Blume, C.; Bock, F.; Bogdanov, A.; Bøggild, H.; Boldizsár, L.; Bombara, M.; Book, J.; Borel, H.; Borissov, A.; Borri, M.; Bossú, F.; Botta, E.; Böttger, S.; Bourjau, C.; Braun-Munzinger, P.; Bregant, M.; Breitner, T.; Broker, T. A.; Browning, T. A.; Broz, M.; Brucken, E. J.; Bruna, E.; Bruno, G. E.; Budnikov, D.; Buesching, H.; Bufalino, S.; Buncic, P.; Busch, O.; Buthelezi, Z.; Butt, J. B.; Buxton, J. T.; Caffarri, D.; Cai, X.; Caines, H.; Calero Diaz, L.; Caliva, A.; Calvo Villar, E.; Camerini, P.; Carena, F.; Carena, W.; Carnesecchi, F.; Castillo Castellanos, J.; Castro, A. J.; Casula, E. A. R.; Ceballos Sanchez, C.; Cepila, J.; Cerello, P.; Cerkala, J.; Chang, B.; Chapeland, S.; Chartier, M.; Charvet, J. L.; Chattopadhyay, S.; Chattopadhyay, S.; Chelnokov, V.; Cherney, M.; Cheshkov, C.; Cheynis, B.; Chibante Barroso, V.; Chinellato, D. D.; Cho, S.; Chochula, P.; Choi, K.; Chojnacki, M.; Choudhury, S.; Christakoglou, P.; Christensen, C. H.; Christiansen, P.; Chujo, T.; Chung, S. U.; Cicalo, C.; Cifarelli, L.; Cindolo, F.; Cleymans, J.; Colamaria, F.; Colella, D.; Collu, A.; Colocci, M.; Conesa Balbastre, G.; Conesa del Valle, Z.; Connors, M. E.; Contreras, J. G.; Cormier, T. M.; Corrales Morales, Y.; Cortés Maldonado, I.; Cortese, P.; Cosentino, M. R.; Costa, F.; Crochet, P.; Cruz Albino, R.; Cuautle, E.; Cunqueiro, L.; Dahms, T.; Dainese, A.; Danu, A.; Das, D.; Das, I.; Das, S.; Dash, A.; Dash, S.; De, S.; De Caro, A.; de Cataldo, G.; de Conti, C.; de Cuveland, J.; De Falco, A.; De Gruttola, D.; De Marco, N.; De Pasquale, S.; Deisting, A.; Deloff, A.; Dénes, E.; Deplano, C.; Dhankher, P.; Di Bari, D.; Di Mauro, A.; Di Nezza, P.; Diaz Corchero, M. A.; Dietel, T.; Dillenseger, P.; Divià, R.; Djuvsland, Ø.; Dobrin, A.; Domenicis Gimenez, D.; Dönigus, B.; Dordic, O.; Drozhzhova, T.; Dubey, A. K.; Dubla, A.; Ducroux, L.; Dupieux, P.; Ehlers, R. J.; Elia, D.; Engel, H.; Epple, E.; Erazmus, B.; Erdemir, I.; Erhardt, F.; Espagnon, B.; Estienne, M.; Esumi, S.; Eum, J.; Evans, D.; Evdokimov, S.; Eyyubova, G.; Fabbietti, L.; Fabris, D.; Faivre, J.; Fantoni, A.; Fasel, M.; Feldkamp, L.; Feliciello, A.; Feofilov, G.; Ferencei, J.; Fernández Téllez, A.; Ferreiro, E. G.; Ferretti, A.; Festanti, A.; Feuillard, V. J. G.; Figiel, J.; Figueredo, M. A. S.; Filchagin, S.; Finogeev, D.; Fionda, F. M.; Fiore, E. M.; Fleck, M. G.; Floris, M.; Foertsch, S.; Foka, P.; Fokin, S.; Fragiacomo, E.; Francescon, A.; Frankenfeld, U.; Fuchs, U.; Furget, C.; Furs, A.; Fusco Girard, M.; Gaardhøje, J. J.; Gagliardi, M.; Gago, A. M.; Gallio, M.; Gangadharan, D. R.; Ganoti, P.; Gao, C.; Garabatos, C.; Garcia-Solis, E.; Gargiulo, C.; Gasik, P.; Gauger, E. F.; Germain, M.; Gheata, A.; Gheata, M.; Ghosh, P.; Ghosh, S. K.; Gianotti, P.; Giubellino, P.; Giubilato, P.; Gladysz-Dziadus, E.; Glässel, P.; Goméz Coral, D. M.; Gomez Ramirez, A.; Gonzalez, V.; González-Zamora, P.; Gorbunov, S.; Görlich, L.; Gotovac, S.; Grabski, V.; Grachov, O. A.; Graczykowski, L. K.; Graham, K. L.; Grelli, A.; Grigoras, A.; Grigoras, C.; Grigoriev, V.; Grigoryan, A.; Grigoryan, S.; Grinyov, B.; Grion, N.; Gronefeld, J. M.; Grosse-Oetringhaus, J. F.; Grossiord, J.-Y.; Grosso, R.; Guber, F.; Guernane, R.; Guerzoni, B.; Gulbrandsen, K.; Gunji, T.; Gupta, A.; Gupta, R.; Haake, R.; Haaland, Ø.; Hadjidakis, C.; Haiduc, M.; Hamagaki, H.; Hamar, G.; Harris, J. W.; Harton, A.; Hatzifotiadou, D.; Hayashi, S.; Heckel, S. T.; Heide, M.; Helstrup, H.; Herghelegiu, A.; Herrera Corral, G.; Hess, B. A.; Hetland, K. F.; Hillemanns, H.; Hippolyte, B.; Hosokawa, R.; Hristov, P.; Huang, M.; Humanic, T. J.; Hussain, N.; Hussain, T.; Hutter, D.; Hwang, D. S.; Ilkaev, R.; Inaba, M.; Ippolitov, M.; Irfan, M.; Ivanov, M.; Ivanov, V.; Izucheev, V.; Jacobs, P. M.; Jadhav, M. B.; Jadlovska, S.; Jadlovsky, J.; Jahnke, C.; Jakubowska, M. J.; Jang, H. J.; Janik, M. A.; Jayarathna, P. H. S. Y.; Jena, C.; Jena, S.; Jimenez Bustamante, R. T.; Jones, P. G.; Jung, H.; Jusko, A.; Kalinak, P.; Kalweit, A.; Kamin, J.; Kang, J. H.; Kaplin, V.; Kar, S.; Karasu Uysal, A.; Karavichev, O.; Karavicheva, T.; Karayan, L.; Karpechev, E.; Kebschull, U.; Keidel, R.; Keijdener, D. L. D.; Keil, M.; Mohisin Khan, M.; Khan, P.; Khan, S. A.; Khanzadeev, A.; Kharlov, Y.; Kileng, B.; Kim, D. W.; Kim, D. J.; Kim, D.; Kim, H.; Kim, J. S.; Kim, M.; Kim, M.; Kim, S.; Kim, T.; Kirsch, S.; Kisel, I.; Kiselev, S.; Kisiel, A.; Kiss, G.; Klay, J. L.; Klein, C.; Klein, J.; Klein-Bösing, C.; Klewin, S.; Kluge, A.; Knichel, M. L.; Knospe, A. G.; Kobayashi, T.; Kobdaj, C.; Kofarago, M.; Kollegger, T.; Kolojvari, A.; Kondratiev, V.; Kondratyeva, N.; Kondratyuk, E.; Konevskikh, A.; Kopcik, M.; Kour, M.; Kouzinopoulos, C.; Kovalenko, O.; Kovalenko, V.; Kowalski, M.; Koyithatta Meethaleveedu, G.; Králik, I.; Kravčáková, A.; Kretz, M.; Krivda, M.; Krizek, F.; Kryshen, E.; Krzewicki, M.; Kubera, A. M.; Kučera, V.; Kuhn, C.; Kuijer, P. G.; Kumar, A.; Kumar, J.; Kumar, L.; Kumar, S.; Kurashvili, P.; Kurepin, A.; Kurepin, A. B.; Kuryakin, A.; Kweon, M. J.; Kwon, Y.; La Pointe, S. L.; La Rocca, P.; Ladron de Guevara, P.; Lagana Fernandes, C.; Lakomov, I.; Langoy, R.; Lara, C.; Lardeux, A.; Lattuca, A.; Laudi, E.; Lea, R.; Leardini, L.; Lee, G. R.; Lee, S.; Lehas, F.; Lemmon, R. C.; Lenti, V.; Leogrande, E.; León Monzón, I.; León Vargas, H.; Leoncino, M.; Lévai, P.; Li, S.; Li, X.; Lien, J.; Lietava, R.; Lindal, S.; Lindenstruth, V.; Lippmann, C.; Lisa, M. A.; Ljunggren, H. M.; Lodato, D. F.; Loenne, P. I.; Loginov, V.; Loizides, C.; Lopez, X.; López Torres, E.; Lowe, A.; Luettig, P.; Lunardon, M.; Luparello, G.; Maevskaya, A.; Mager, M.; Mahajan, S.; Mahmood, S. M.; Maire, A.; Majka, R. D.; Malaev, M.; Maldonado Cervantes, I.; Malinina, L.; Mal'Kevich, D.; Malzacher, P.; Mamonov, A.; Manko, V.; Manso, F.; Manzari, V.; Marchisone, M.; Mareš, J.; Margagliotti, G. V.; Margotti, A.; Margutti, J.; Marín, A.; Markert, C.; Marquard, M.; Martin, N. A.; Martin Blanco, J.; Martinengo, P.; Martínez, M. I.; Martínez García, G.; Martinez Pedreira, M.; Mas, A.; Masciocchi, S.; Masera, M.; Masoni, A.; Massacrier, L.; Mastroserio, A.; Matyja, A.; Mayer, C.; Mazer, J.; Mazzoni, M. A.; Mcdonald, D.; Meddi, F.; Melikyan, Y.; Menchaca-Rocha, A.; Meninno, E.; Mercado Pérez, J.; Meres, M.; Miake, Y.; Mieskolainen, M. M.; Mikhaylov, K.; Milano, L.; Milosevic, J.; Minervini, L. M.; Mischke, A.; Mishra, A. N.; Miśkowiec, D.; Mitra, J.; Mitu, C. M.; Mohammadi, N.; Mohanty, B.; Molnar, L.; Montaño Zetina, L.; Montes, E.; Moreira De Godoy, D. A.; Moreno, L. A. P.; Moretto, S.; Morreale, A.; Morsch, A.; Muccifora, V.; Mudnic, E.; Mühlheim, D.; Muhuri, S.; Mukherjee, M.; Mulligan, J. D.; Munhoz, M. G.; Munzer, R. H.; Murray, S.; Musa, L.; Musinsky, J.; Naik, B.; Nair, R.; Nandi, B. K.; Nania, R.; Nappi, E.; Naru, M. U.; Natal da Luz, H.; Nattrass, C.; Nayak, K.; Nayak, T. K.; Nazarenko, S.; Nedosekin, A.; Nellen, L.; Ng, F.; Nicassio, M.; Niculescu, M.; Niedziela, J.; Nielsen, B. S.; Nikolaev, S.; Nikulin, S.; Nikulin, V.; Noferini, F.; Nomokonov, P.; Nooren, G.; Noris, J. C. C.; Norman, J.; Nyanin, A.; Nystrand, J.; Oeschler, H.; Oh, S.; Oh, S. K.; Ohlson, A.; Okatan, A.; Okubo, T.; Olah, L.; Oleniacz, J.; Oliveira Da Silva, A. C.; Oliver, M. H.; Onderwaater, J.; Oppedisano, C.; Orava, R.; Ortiz Velasquez, A.; Oskarsson, A.; Otwinowski, J.; Oyama, K.; Ozdemir, M.; Pachmayer, Y.; Pagano, P.; Paić, G.; Pal, S. K.; Pan, J.; Pandey, A. K.; Papcun, P.; Papikyan, V.; Pappalardo, G. S.; Pareek, P.; Park, W. J.; Parmar, S.; Passfeld, A.; Paticchio, V.; Patra, R. N.; Paul, B.; Peitzmann, T.; Pereira Da Costa, H.; Pereira De Oliveira Filho, E.; Peresunko, D.; Pérez Lara, C. E.; Perez Lezama, E.; Peskov, V.; Pestov, Y.; Petráček, V.; Petrov, V.; Petrovici, M.; Petta, C.; Piano, S.; Pikna, M.; Pillot, P.; Pinazza, O.; Pinsky, L.; Piyarathna, D. B.; Płoskoń, M.; Planinic, M.; Pluta, J.; Pochybova, S.; Podesta-Lerma, P. L. M.; Poghosyan, M. G.; Polichtchouk, B.; Poljak, N.; Poonsawat, W.; Pop, A.; Porteboeuf-Houssais, S.; Porter, J.; Pospisil, J.; Prasad, S. K.; Preghenella, R.; Prino, F.; Pruneau, C. A.; Pshenichnov, I.; Puccio, M.; Puddu, G.; Pujahari, P.; Punin, V.; Putschke, J.; Qvigstad, H.; Rachevski, A.; Raha, S.; Rajput, S.; Rak, J.; Rakotozafindrabe, A.; Ramello, L.; Rami, F.; Raniwala, R.; Raniwala, S.; Räsänen, S. S.; Rascanu, B. T.; Rathee, D.; Read, K. F.; Redlich, K.; Reed, R. J.; Rehman, A.; Reichelt, P.; Reidt, F.; Ren, X.; Renfordt, R.; Reolon, A. R.; Reshetin, A.; Revol, J.-P.; Reygers, K.; Riabov, V.; Ricci, R. A.; Richert, T.; Richter, M.; Riedler, P.; Riegler, W.; Riggi, F.; Ristea, C.; Rocco, E.; Rodríguez Cahuantzi, M.; Rodriguez Manso, A.; Røed, K.; Rogochaya, E.; Rohr, D.; Röhrich, D.; Romita, R.; Ronchetti, F.; Ronflette, L.; Rosnet, P.; Rossi, A.; Roukoutakis, F.; Roy, A.; Roy, C.; Roy, P.; Rubio Montero, A. J.; Rui, R.; Russo, R.; Ryabinkin, E.; Ryabov, Y.; Rybicki, A.; Sadovsky, S.; Šafařík, K.; Sahlmuller, B.; Sahoo, P.; Sahoo, R.; Sahoo, S.; Sahu, P. K.; Saini, J.; Sakai, S.; Saleh, M. A.; Salzwedel, J.; Sambyal, S.; Samsonov, V.; Šándor, L.; Sandoval, A.; Sano, M.; Sarkar, D.; Scapparone, E.; Scarlassara, F.; Schiaua, C.; Schicker, R.; Schmidt, C.; Schmidt, H. R.; Schuchmann, S.; Schukraft, J.; Schulc, M.; Schuster, T.; Schutz, Y.; Schwarz, K.; Schweda, K.; Scioli, G.; Scomparin, E.; Scott, R.; Šefčík, M.; Seger, J. E.; Sekiguchi, Y.; Sekihata, D.; Selyuzhenkov, I.; Senosi, K.; Senyukov, S.; Serradilla, E.; Sevcenco, A.; Shabanov, A.; Shabetai, A.; Shadura, O.; Shahoyan, R.; Shangaraev, A.; Sharma, A.; Sharma, M.; Sharma, M.; Sharma, N.; Shigaki, K.; Shtejer, K.; Sibiriak, Y.; Siddhanta, S.; Sielewicz, K. M.; Siemiarczuk, T.; Silvermyr, D.; Silvestre, C.; Simatovic, G.; Simonetti, G.; Singaraju, R.; Singh, R.; Singha, S.; Singhal, V.; Sinha, B. C.; Sinha, T.; Sitar, B.; Sitta, M.; Skaali, T. B.; Slupecki, M.; Smirnov, N.; Snellings, R. J. M.; Snellman, T. W.; Søgaard, C.; Song, J.; Song, M.; Song, Z.; Soramel, F.; Sorensen, S.; Sozzi, F.; Spacek, M.; Spiriti, E.; Sputowska, I.; Spyropoulou-Stassinaki, M.; Stachel, J.; Stan, I.; Stefanek, G.; Stenlund, E.; Steyn, G.; Stiller, J. H.; Stocco, D.; Strmen, P.; Suaide, A. A. P.; Sugitate, T.; Suire, C.; Suleymanov, M.; Suljic, M.; Sultanov, R.; Šumbera, M.; Szabo, A.; Szanto de Toledo, A.; Szarka, I.; Szczepankiewicz, A.; Szymanski, M.; Tabassam, U.; Takahashi, J.; Tambave, G. J.; Tanaka, N.; Tangaro, M. A.; Tarhini, M.; Tariq, M.; Tarzila, M. G.; Tauro, A.; Tejeda Muñoz, G.; Telesca, A.; Terasaki, K.; Terrevoli, C.; Teyssier, B.; Thäder, J.; Thomas, D.; Tieulent, R.; Timmins, A. R.; Toia, A.; Trogolo, S.; Trombetta, G.; Trubnikov, V.; Trzaska, W. H.; Tsuji, T.; Tumkin, A.; Turrisi, R.; Tveter, T. S.; Ullaland, K.; Uras, A.; Usai, G. L.; Utrobicic, A.; Vajzer, M.; Vala, M.; Valencia Palomo, L.; Vallero, S.; Van Der Maarel, J.; Van Hoorne, J. W.; van Leeuwen, M.; Vanat, T.; Vande Vyvre, P.; Varga, D.; Vargas, A.; Vargyas, M.; Varma, R.; Vasileiou, M.; Vasiliev, A.; Vauthier, A.; Vechernin, V.; Veen, A. M.; Veldhoen, M.; Velure, A.; Venaruzzo, M.; Vercellin, E.; Vergara Limón, S.; Vernet, R.; Verweij, M.; Vickovic, L.; Viesti, G.; Viinikainen, J.; Vilakazi, Z.; Villalobos Baillie, O.; Villatoro Tello, A.; Vinogradov, A.; Vinogradov, L.; Vinogradov, Y.; Virgili, T.; Vislavicius, V.; Viyogi, Y. P.; Vodopyanov, A.; Völkl, M. A.; Voloshin, K.; Voloshin, S. A.; Volpe, G.; von Haller, B.; Vorobyev, I.; Vranic, D.; Vrláková, J.; Vulpescu, B.; Vyushin, A.; Wagner, B.; Wagner, J.; Wang, H.; Wang, M.; Watanabe, D.; Watanabe, Y.; Weber, M.; Weber, S. G.; Weiser, D. F.; Wessels, J. P.; Westerhoff, U.; Whitehead, A. M.; Wiechula, J.; Wikne, J.; Wilde, M.; Wilk, G.; Wilkinson, J.; Williams, M. C. S.; Windelband, B.; Winn, M.; Yaldo, C. G.; Yang, H.; Yang, P.; Yano, S.; Yasar, C.; Yin, Z.; Yokoyama, H.; Yoo, I.-K.; Yoon, J. H.; Yurchenko, V.; Yushmanov, I.; Zaborowska, A.; Zaccolo, V.; Zaman, A.; Zampolli, C.; Zanoli, H. J. C.; Zaporozhets, S.; Zardoshti, N.; Zarochentsev, A.; Závada, P.; Zaviyalov, N.; Zbroszczyk, H.; Zgura, I. S.; Zhalov, M.; Zhang, H.; Zhang, X.; Zhang, Y.; Zhang, C.; Zhang, Z.; Zhao, C.; Zhigareva, N.; Zhou, D.; Zhou, Y.; Zhou, Z.; Zhu, H.; Zhu, J.; Zichichi, A.; Zimmermann, A.; Zimmermann, M. B.; Zinovjev, G.; Zyzak, M.

    2016-03-01

    The centrality dependence of the charged-particle pseudorapidity density measured with ALICE in Pb-Pb collisions at √{sNN} = 2.76 TeV over a broad pseudorapidity range is presented. This Letter extends the previous results reported by ALICE to more peripheral collisions. No strong change of the overall shape of charged-particle pseudorapidity density distributions with centrality is observed, and when normalised to the number of participating nucleons in the collisions, the evolution over pseudorapidity with centrality is likewise small. The broad pseudorapidity range (- 3.5 < η < 5) allows precise estimates of the total number of produced charged particles which we find to range from 162 ± 22(syst.) to 17170 ± 770(syst.) in 80-90% and 0-5% central collisions, respectively. The total charged-particle multiplicity is seen to approximately scale with the number of participating nucleons in the collision. This suggests that hard contributions to the charged-particle multiplicity are limited. The results are compared to models which describe dNch / dη at mid-rapidity in the most central Pb-Pb collisions and it is found that these models do not capture all features of the distributions.

  7. HTCC: Broad Range Inhibitor of Coronavirus Entry

    PubMed Central

    Milewska, Aleksandra; Kaminski, Kamil; Ciejka, Justyna; Kosowicz, Katarzyna; Zeglen, Slawomir; Wojarski, Jacek; Nowakowska, Maria; Szczubiałka, Krzysztof; Pyrc, Krzysztof

    2016-01-01

    To date, six human coronaviruses have been known, all of which are associated with respiratory infections in humans. With the exception of the highly pathogenic SARS and MERS coronaviruses, human coronaviruses (HCoV-NL63, HCoV-OC43, HCoV-229E, and HCoV-HKU1) circulate worldwide and typically cause the common cold. In most cases, infection with these viruses does not lead to severe disease, although acute infections in infants, the elderly, and immunocompromised patients may progress to severe disease requiring hospitalization. Importantly, no drugs against human coronaviruses exist, and only supportive therapy is available. Previously, we proposed the cationically modified chitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and its hydrophobically-modified derivative (HM-HTCC) as potent inhibitors of the coronavirus HCoV-NL63. Here, we show that HTCC inhibits interaction of a virus with its receptor and thus blocks the entry. Further, we demonstrate that HTCC polymers with different degrees of substitution act as effective inhibitors of all low-pathogenic human coronaviruses. PMID:27249425

  8. Sequencing of 16S rRNA Gene: A Rapid Tool for Identification of Bacillus anthracis

    PubMed Central

    Whitney, Anne M.; Mayer, Leonard W.; Morey, Roger; Steigerwalt, Arnold; Boras, Ariana; Weyant, Robin S.; Popovic, Tanja

    2002-01-01

    In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA generom these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA gene seqs fuences that differed from each other at 1 to 8 positions (0.06% to 0.5%). All 86 B. anthracis had an identical 16S gene sequence, designated type 6; 16S type 10 was seen in all B. thuringiensis strains; six other 16S types were found among the 10 B. cereus strains. This report describes the first demonstration of an exclusive association of a distinct 16S sequence with B. anthracis. Consequently, we were able to rapidly identify suspected isolates and to detect the B. anthracis 16S rRNA gene directly from culture-negative clinical specimens from seven patients with laboratory-confirmed anthrax. PMID:12396926

  9. Y{sub 3}Fe{sub 5}O{sub 12} spin pumping for quantitative understanding of pure spin transport and spin Hall effect in a broad range of materials (invited)

    SciTech Connect

    Du, Chunhui; Wang, Hailong; Hammel, P. Chris; Yang, Fengyuan

    2015-05-07

    Using Y{sub 3}Fe{sub 5}O{sub 12} (YIG) thin films grown by our sputtering technique, we study dynamic spin transport in nonmagnetic, ferromagnetic, and antiferromagnetic (AF) materials by ferromagnetic resonance spin pumping. From both inverse spin Hall effect and damping enhancement, we determine the spin mixing conductance and spin Hall angle in many metals. Surprisingly, we observe robust spin conduction in AF insulators excited by an adjacent YIG at resonance. This demonstrates that YIG spin pumping is a powerful and versatile tool for understanding spin Hall physics, spin-orbit coupling, and magnetization dynamics in a broad range of materials.

  10. Identification of nontuberculous mycobacteria using multilocous sequence analysis of 16S rRNA, hsp65, and rpoB.

    PubMed

    Kim, Si Hyun; Shin, Jeong Hwan

    2017-02-23

    The isolation of nontuberculous mycobacteria (NTM) from clinical specimens has increased, and they now are considered significant opportunistic pathogens. The aims of this study were to develop a database and interpretive criteria for identifying individual species. In addition, using clinical isolates, we evaluated the clinical usefulness of 16S rRNA, hsp65, and rpoB as target genes for this method. The sequences of NTM for 16S rRNA, hsp65, and rpoB were collected from GenBank and checked by manual inspection. Clinical isolates collected between 2005 and 2010 were used for DNA extraction, polymerase chain reaction, and sequencing of these three genes. We constructed a database for the genes and evaluated the clinical utility of multilocus sequence analysis (MLSA) using 109 clinical isolates. A total 131, 130, and 122 sequences were collected from GenBank for 16S rRNA, hsp65, and rpoB, respectively. The percent similarities of the three genes ranged from 96.57% to 100% for the 16S rRNA gene, 89.27% to 100% for hsp65, and 92.71% to 100% for rpoB. When we compared the sequences of 109 clinical strains with those of the database, the rates of species-level identification were 71.3%, 86.79%, and 81.55% with 16S rRNA, hsp65, and rpoB, respectively. We could identify 97.25% of the isolates to the species level when we used MLSA. There were significant differences among the utilities of the three genes for species identification. The MLSA technique would be helpful for identification of NTM. © 2017 Wiley Periodicals, Inc.

  11. The use of 16S and 16S-23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes.

    PubMed

    Jeng, R S; Svircev, A M; Myers, A L; Beliaeva, L; Hunter, D M; Hubbes, M

    2001-02-01

    The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consuming and tedious process for the plant pathologist. The paper provides a simple "one-step" protocol that uses the polymerase chain reaction (PCR) to amplify intergenic spacer regions between 16S and 23S genes and a portion of 16S gene in the prokaryotic rRNA genetic loci. Amplified 16S rDNA, and restriction fragment length polymorphisms (RFLP) following EcoRI digestion produced band patterns that readily distinguished between the plant pathogen Erwinia amylovora (causal agent of fire blight in pear and apple) and the orchard epiphyte Pantoea agglomerans (formerly E. herbicola). The amplified DNA patterns of 16S-23S spacer regions may be used to differentiate E. amylovora at the intraspecies level. Isolates of E. amylovora obtained from raspberries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands. In addition, the size of the 16S-23S spacer region differs between Pseudomonas syringae and Pseudomonas fluorescens. The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes.

  12. Hosts, distribution and genetic divergence (16S rDNA) of Amblyomma dubitatum (Acari: Ixodidae).

    PubMed

    Nava, Santiago; Venzal, José M; Labruna, Marcelo B; Mastropaolo, Mariano; González, Enrique M; Mangold, Atilio J; Guglielmone, Alberto A

    2010-08-01

    We supply information about hosts and distribution of Amblyomma dubitatum. In addition, we carry out an analysis of genetic divergence among specimens of A. dubitatum from different localities and with respect to other Neotropical Amblyomma species, using sequences of 16S rDNA gene. Although specimens of A. dubitatum were collected on several mammal species as cattle horse, Tapirus terrestris, Mazama gouazoubira, Tayassu pecari, Sus scrofa, Cerdocyon thous, Myocastor coypus, Allouata caraya, Glossophaga soricina and man, most records of immature and adult stages of A. dubitatum were made on Hydrochoerus hydrochaeris, making this rodent the principal host for all parasitic stages of this ticks. Cricetidae rodents (Lundomys molitor, Scapteromys tumidus), opossums (Didelphis albiventris) and vizcacha (Lagostomus maximus) also were recorded as hosts for immature stages. All findings of A. dubitatum correspond to localities of Argentina, Brazil, Paraguay and Uruguay, and they were concentrated in the Biogeographical provinces of Pampa, Chaco, Cerrado, Brazilian Atlantic Forest, Parana Forest and Araucaria angustifolia Forest. The distribution of A. dubitatum is narrower than that of its principal host, therefore environmental variables rather than hosts determine the distributional ranges of this tick. The intraspecific genetic divergence among 16S rDNA sequences of A. dubitatum ticks collected in different localities from Argentina, Brazil and Uruguay was in all cases lower than 0.8%, whereas the differences with the remaining Amblyomma species included in the analysis were always bigger than 6.8%. Thus, the taxonomic status of A. dubitatum along its distribution appears to be certain at the specific level.

  13. Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin

    PubMed Central

    Chahine, Sarah; Okafor, Darius; Ong, Ana C.; Maybank, Rosslyn; Kwak, Yoon I.; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

    2015-01-01

    16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. PMID:26537447

  14. Bacterial Diversity Studies Using the 16S rRNA Gene Provide a Powerful Research-Based Curriculum for Molecular Biology Laboratory

    PubMed Central

    BOOMER, SARAH M.; LODGE, DANIEL P.; DUTTON, BRYAN E.

    2002-01-01

    We have developed a ten-week curriculum for molecular biology that uses 16S ribosomal RNA genes to characterize and compare novel bacteria from hot spring communities in Yellowstone National Park. The 16S rRNA approach bypasses selective culture-based methods. Our molecular biology course offered the opportunity for students to learn broadly applicable methods while contributing to a long-term research project. Specifically, students isolated and characterized clones that contained novel 16S rRNA inserts using restriction enzyme, DNA sequencing, and computer-based phylogenetic methods. In both classes, students retrieved novel bacterial 16S rRNA genes, several of which were most similar to Green Nonsulfur bacterial isolates. During class, we evaluated student performance and mastery of skills and concepts using quizzes, formal lab notebooks, and a broad project assignment. For this report, we also assessed student performance alongside data quality and discussed the significance, our goal being to improve both research and teaching methods. PMID:23653546

  15. Monitoring Precursor 16S rRNAs of Acinetobacter spp. in Activated Sludge Wastewater Treatment Systems

    PubMed Central

    Oerther, Daniel B.; Pernthaler, Jakob; Schramm, Andreas; Amann, Rudolf; Raskin, Lutgarde

    2000-01-01

    Recently, Cangelosi and Brabant used oligonucleotide probes targeting the precursor 16S rRNA of Escherichia coli to demonstrate that the levels of precursor rRNA were more sensitive to changes in growth phase than the levels of total rRNA (G. A. Cangelosi and W. H. Brabant, J. Bacteriol. 179:4457–4463, 1997). In order to measure changes in the levels of precursor rRNA in activated sludge systems, we designed oligonucleotide probes targeting the 3′ region of the precursor 16S rRNA of Acinetobacter spp. We used these probes to monitor changes in the level of precursor 16S rRNA during batch growth of Acinetobacter spp. in Luria-Bertani (LB) medium, filtered wastewater, and in lab- and full-scale wastewater treatment systems. Consistent with the previous reports for E. coli, results obtained with membrane hybridizations and fluorescence in situ hybridizations with Acinetobacter calcoaceticus grown in LB medium showed a more substantial and faster increase in precursor 16S rRNA levels compared to the increase in total 16S rRNA levels during exponential growth. Diluting an overnight culture of A. calcoaceticus grown in LB medium with filtered wastewater resulted in a pattern of precursor 16S rRNA levels that appeared to follow diauxic growth. In addition, fluorescence in situ hybridizations with oligonucleotide probes targeting total 16S rRNA and precursor 16S rRNA showed that individual cells of A. calcoaceticus expressed highly variable levels of precursor 16S rRNA when adapting from LB medium to filtered sewage. Precursor 16S rRNA levels of Acinetobacter spp. transiently increased when activated sludge was mixed with influent wastewater in lab- and full-scale wastewater treatment systems. These results suggest that Acinetobacter spp. experience a change in growth activity within wastewater treatment systems. PMID:10788395

  16. Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.).

    PubMed

    Ormeño-Orrillo, Ernesto; Menna, Pâmela; Almeida, Luiz Gonzaga P; Ollero, Francisco Javier; Nicolás, Marisa Fabiana; Pains Rodrigues, Elisete; Shigueyoshi Nakatani, Andre; Silva Batista, Jesiane Stefânia; Oliveira Chueire, Ligia Maria; Souza, Rangel Celso; Ribeiro Vasconcelos, Ana Tereza; Megías, Manuel; Hungria, Mariangela; Martínez-Romero, Esperanza

    2012-12-27

    Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 are α-Proteobacteria that establish nitrogen-fixing symbioses with a range of legume hosts. These strains are broadly used in commercial inoculants for application to common bean (Phaseolus vulgaris) in South America and Africa. Both strains display intrinsic resistance to several abiotic stressful conditions such as low soil pH and high temperatures, which are common in tropical environments, and to several antimicrobials, including pesticides. The genetic determinants of these interesting characteristics remain largely unknown. Genome sequencing revealed that CIAT 899 and PRF 81 share a highly-conserved symbiotic plasmid (pSym) that is present also in Rhizobium leucaenae CFN 299, a rhizobium displaying a similar host range. This pSym seems to have arisen by a co-integration event between two replicons. Remarkably, three distinct nodA genes were found in the pSym, a characteristic that may contribute to the broad host range of these rhizobia. Genes for biosynthesis and modulation of plant-hormone levels were also identified in the pSym. Analysis of genes involved in stress response showed that CIAT 899 and PRF 81 are well equipped to cope with low pH, high temperatures and also with oxidative and osmotic stresses. Interestingly, the genomes of CIAT 899 and PRF 81 had large numbers of genes encoding drug-efflux systems, which may explain their high resistance to antimicrobials. Genome analysis also revealed a wide array of traits that may allow these strains to be successful rhizosphere colonizers, including surface polysaccharides, uptake transporters and catabolic enzymes for nutrients, diverse iron-acquisition systems, cell wall-degrading enzymes, type I and IV pili, and novel T1SS and T5SS secreted adhesins. Availability of the complete genome sequences of CIAT 899 and PRF 81 may be exploited in further efforts to understand the interaction of tropical rhizobia with common bean and other legume hosts.

  17. Investigation of the koala (Phascolarctos cinereus) hindgut microbiome via 16S pyrosequencing.

    PubMed

    Barker, Christopher J; Gillett, Amber; Polkinghorne, Adam; Timms, Peter

    2013-12-27

    As a dietary source, the foliage of Eucalyptus spp. is low in available protein and carbohydrate while containing polyphenolic compounds that interfere with enzymatic digestion. To overcome this, the koala (Phascolarctos cinereus) has evolved a range of anatomical and physiological adaptations to assist with digestion and absorption of nutrients from this food source. Microbial fermentation of partially digested eucalyptus leaves is thought to be critical in this process, however, little is known about the composition and diversity of microorganisms that are associated with digestive health in this native species. In this study, we performed 16S rRNA gene pyrosequencing of caecum, colon and faecal pellet samples from two wild, free ranging, Queensland koalas. Our results reveal a highly complex and diverse ecosystem with considerable intra-individual variation. Although samples were dominated by sequences from the Bacteroidetes and Firmicutes phyla there was considerable variation at the genus level. This study is the first non-culture based microbiota analysis, using 454-amplicon pyrosequencing, and provides preliminary data to expand our understanding of the koala hindgut.

  18. Intrageneric structure of the genus Gluconobacter analyzed by the 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    Takahashi, Mai; Yukphan, Pattaraporn; Yamada, Yuzo; Suzuki, Ken-ichiro; Sakane, Takeshi; Nakagawa, Yasuyoshi

    2006-06-01

    Forty-nine strains belonging to the genus Gluconobacter were re-examined with respect to their species identification based on the sequences of the 16S rDNA and 16S-23S rDNA internal transcribed spacer regions (ITS). A phylogenetic tree constructed from the 16S rDNA sequences indicated the presence of five clusters corresponding, respectively, to the major five species of the genus Gluconobacter, namely G. albidus, G. cerinus, G. frateurii, G. oxydans (type species), and G. thailandicus. The type strain of G. asaii, NBRC 3276T (T=type strain) was included in the G. cerinus cluster, which is consistent with the report that G. asaii is a junior subjective synonym of G. cerinus. Existence of the G. albidus, G. cerinus, G. frateurii, G. oxydans, and G. thailandicus clusters was also recognized by the ITS sequence analysis. Both sequence analyses revealed that the G. cerinus and G. frateurii clusters were heterogeneous. The G. cerinus cluster comprised three strains of G. cerinus and one strain of G. frateurii, while the G. frateurii cluster included ten strains of G. frateurii, three of G. cerinus, and eleven of G. oxydans. These results suggest that phenotypic differences among Gluconobacter species are ambiguous and the species definition must be re-evaluated. The 16S rDNA and ITS sequences determined in this study are valuable for the identification and phylogenetic analysis of Gluconobacter species.

  19. Incorporating 16S gene copy number information improves estimates of microbial diversity and abundance.

    PubMed

    Kembel, Steven W; Wu, Martin; Eisen, Jonathan A; Green, Jessica L

    2012-01-01

    The abundance of different SSU rRNA ("16S") gene sequences in environmental samples is widely used in studies of microbial ecology as a measure of microbial community structure and diversity. However, the genomic copy number of the 16S gene varies greatly - from one in many species to up to 15 in some bacteria and to hundreds in some microbial eukaryotes. As a result of this variation the relative abundance of 16S genes in environmental samples can be attributed both to variation in the relative abundance of different organisms, and to variation in genomic 16S copy number among those organisms. Despite this fact, many studies assume that the abundance of 16S gene sequences is a surrogate measure of the relative abundance of the organisms containing those sequences. Here we present a method that uses data on sequences and genomic copy number of 16S genes along with phylogenetic placement and ancestral state estimation to estimate organismal abundances from environmental DNA sequence data. We use theory and simulations to demonstrate that 16S genomic copy number can be accurately estimated from the short reads typically obtained from high-throughput environmental sequencing of the 16S gene, and that organismal abundances in microbial communities are more strongly correlated with estimated abundances obtained from our method than with gene abundances. We re-analyze several published empirical data sets and demonstrate that the use of gene abundance versus estimated organismal abundance can lead to different inferences about community diversity and structure and the identity of the dominant taxa in microbial communities. Our approach will allow microbial ecologists to make more accurate inferences about microbial diversity and abundance based on 16S sequence data.

  20. Two different 16S rRNA genes in a mycobacterial strain.

    PubMed Central

    Ninet, B; Monod, M; Emler, S; Pawlowski, J; Metral, C; Rohner, P; Auckenthaler, R; Hirschel, B

    1996-01-01

    Sequencing of the gene coding for 16S rRNA (16S rDNA) is a well-established method used to identify bacteria, particularly mycobacteria. Unique sequences allow identification of a particular genus and species. If more than one 16S rDNA is present on one mycobacterial genome, their sequences are assumed to be strictly or almost identical. We have isolated a slowly growing Mycobacterium strain, "X", identified by conventional biochemical tests as Mycobacterium terrae. Identification by amplification and direct sequencing of 16S rDNA yielded ambiguous results in two variable regions, suggesting the presence of different copies of the sequenced gene. Total DNA was digested by restriction enzymes and hybridized after Southern blotting to a probe representing about two-thirds of the 16S rDNA. Two copies of 16S rDNA were identified and cloned. By sequencing, the clones were of two different types, A and B, differing in 18 positions. Oligonucleotides specific to each copy of the 16S rDNA were used to distinguish the positions of the two genes observed in the Southern blot. We conclude that Mycobacterium strain "X" has two different copies of 16S rDNA. Variations in the sequence between two copies of 16S rDNA gene have been described in archaeobacteria, but not in mycobacteria. When placed in a phylogenetic tree together with other slowly growing mycobacteria gene A shows a common root with M. terrae, whereas gene B is placed separately. PMID:8880515

  1. A new protocol to detect multiple foodborne pathogens with PCR dipstick DNA chromatography after a six-hour enrichment culture in a broad-range food pathogen enrichment broth.

    PubMed

    Hayashi, Masahiro; Natori, Tatsuya; Kubota-Hayashi, Sayoko; Miyata, Machiko; Ohkusu, Kiyofumi; Kawamoto, Keiko; Kurazono, Hisao; Makino, Souichi; Ezaki, Takayuki

    2013-01-01

    A quick foodborne pathogen screening method after six-hour enrichment culture with a broad-range food pathogen enrichment broth is described. Pathogenic factors of Salmonella enterica, Shigella spp., enteroinvasive Escherichia coli, and enterohemorrhagic E. coli are amplified with a cocktail primer and rapid polymerase chain reaction (PCR), which finishes amplification in 30 min. The PCR amplicon was differentiated with a dipstick DNA chromatography assay in 5-10 min. Starting from a four- to six-hour enrichment culture, this assay was finished within 45 min. Detection sensitivity of this protocol was less than 2.5 CFU/25 g for S. enterica and 3.3 CFU/25 g for enterohemorrhagic E. coli in spiked ground meat experiments.

  2. Centrality evolution of the charged-particle pseudorapidity density over a broad pseudorapidity range in Pb–Pb collisions at sNN=2.76 TeV

    DOE PAGES

    Adam, J.; Adamová, D.; Aggarwal, M. M.; ...

    2016-01-26

    The centrality dependence of the charged-particle pseudorapidity density measured with ALICE in Pb-Pb collisions at √sNN = 2.76 TeV over a broad pseudorapidity range is presented. This Letter extends the previous results reported by ALICE to more peripheral collisions. No strong change of the overall shape of charged-particle pseudorapidity density distributions with centrality is observed, and when normalised to the number of participating nucleons in the collisions, the evolution over pseudorapidity with centrality is likewise small. Broadening the pseudorapidity range (-3.5 < η < 5) allows precise estimates of the total number of produced charged particles which we find tomore » range from 162 ± 22(syst.) to 17170 ± 770(syst.) in 80-90% and 0-5% central collisions, respectively. The total charged-particle multiplicity is seen to approximately scale with the number of participating nucleons in the collision. This suggests that hard contributions to the charged-particle multiplicity are limited. Our results are compared to models which describe dNch/dη at mid-rapidity in the most central Pb-Pb collisions and it is found that these models do not capture all features of the distributions.« less

  3. Tsukamurella tyrosinosolvens intravascular catheter infection identified using 16S ribosomal DNA sequencing.

    PubMed

    Sheridan, Elizabeth A S; Warwick, Simon; Chan, Anthony; Dall'Antonia, Martino; Koliou, Maria; Sefton, Armine

    2003-03-01

    Cultures of blood from a hemodialysis line repeatedly yielded a gram-positive rod. The organism was identified as Tsukamurella tyrosinosolvens by 16S ribosomal DNA sequencing, and the patient was treated successfully by removal of the line.

  4. Evaluation of 16S rDNA-based community profiling for human microbiome research.

    PubMed

    2012-01-01

    The Human Microbiome Project will establish a reference data set for analysis of the microbiome of healthy adults by surveying multiple body sites from 300 people and generating data from over 12,000 samples. To characterize these samples, the participating sequencing centers evaluated and adopted 16S rDNA community profiling protocols for ABI 3730 and 454 FLX Titanium sequencing. In the course of establishing protocols, we examined the performance and error characteristics of each technology, and the relationship of sequence error to the utility of 16S rDNA regions for classification- and OTU-based analysis of community structure. The data production protocols used for this work are those used by the participating centers to produce 16S rDNA sequence for the Human Microbiome Project. Thus, these results can be informative for interpreting the large body of clinical 16S rDNA data produced for this project.

  5. Probing the structure of 16 S ribosomal RNA from Bacillus brevis.

    PubMed

    Kop, J; Kopylov, A M; Magrum, L; Siegel, R; Gupta, R; Woese, C R; Noller, H F

    1984-12-25

    A majority (approximately 89%) of the nucleotide sequence of Bacillus brevis 16 S rRNA has been determined by a combination of RNA sequencing methods. Several experimental approaches have been used to probe its structure, including (a) partial RNase digestion of 30 S ribosomal subunits, followed by two-dimensional native/denatured gel electrophoresis, in which base-paired fragments were directly identified; (b) identification of positions susceptible to cleavage by RNase A and RNase T1 in 30 S subunits; (c) sites of attack by cobra venom RNase on naked 16 S rRNA; and (d) nucleotides susceptible to attack by bisulfite in 16 S rRNA. These data are discussed with respect to a secondary structure model for B. brevis 16 S rRNA derived by comparative sequence analysis.

  6. Phylogenetic positions of Clostridium novyi and Clostridium haemolyticum based on 16S rDNA sequences.

    PubMed

    Sasaki, Y; Takikawa, N; Kojima, A; Norimatsu, M; Suzuki, S; Tamura, Y

    2001-05-01

    The partial sequences (1465 bp) of the 16S rDNA of Clostridium novyi types A, B and C and Clostridium haemolyticum were determined. C. novyi types A, B and C and C. haemolyticum clustered with Clostridium botulinum types C and D. Moreover, the 16S rDNA sequences of C. novyi type B strains and C. haemolyticum strains were completely identical; they differed by 1 bp (level of similarity > 99.9%) from that of C. novyi type C, they were 98.7% homologous to that of C. novyi type A (relative positions 28-1520 of the Escherichia coli 16S rDNA sequence) and they exhibited a higher similarity to the 16S rDNA sequence of C. botulinum types D and C than to that of C. novyi type A. These results suggest that C. novyi types B and C and C. haemolyticum may be one independent species generated from the same phylogenetic origin.

  7. A Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife.

    PubMed

    Razzauti, Maria; Galan, Maxime; Bernard, Maria; Maman, Sarah; Klopp, Christophe; Charbonnel, Nathalie; Vayssier-Taussat, Muriel; Eloit, Marc; Cosson, Jean-François

    2015-01-01

    Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq) and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations. We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq). In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454). In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles. We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each individual reservoir, with subsequent derivation of bacterial prevalence

  8. A Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife

    PubMed Central

    Razzauti, Maria; Galan, Maxime; Bernard, Maria; Maman, Sarah; Klopp, Christophe; Charbonnel, Nathalie; Vayssier-Taussat, Muriel; Eloit, Marc; Cosson, Jean-François

    2015-01-01

    Background Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq) and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations. Methodology/Principal Findings We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq). In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454). In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles. Conclusions/Significance We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each

  9. Use of 16S Ribosomal RNA Sequences to Infer Relationships among Archaebacteria.

    DTIC Science & Technology

    1987-04-16

    FIELD GROUP SUB-GROUP Archaebacteria; Eubacteria ; Eukaryotes; 16S Ribosomal RNA; 08 I Phylogeny; rRNA; RNA Sequencing; Molecular Clock; Urkingdoms; r...16S rRNA data were used to infer the relat onships among the archaebacteria, and of the archaebacteria to the eubacteria and eukaryotes. ur programs for...been published (1, 2, 16, 18). The analyses render untenable the suggestions of Lake and colleagues (Lake et al., 1985) that the eubacteria derive from

  10. Incorporating 16S Gene Copy Number Information Improves Estimates of Microbial Diversity and Abundance

    PubMed Central

    Kembel, Steven W.; Wu, Martin; Eisen, Jonathan A.; Green, Jessica L.

    2012-01-01

    The abundance of different SSU rRNA (“16S”) gene sequences in environmental samples is widely used in studies of microbial ecology as a measure of microbial community structure and diversity. However, the genomic copy number of the 16S gene varies greatly – from one in many species to up to 15 in some bacteria and to hundreds in some microbial eukaryotes. As a result of this variation the relative abundance of 16S genes in environmental samples can be attributed both to variation in the relative abundance of different organisms, and to variation in genomic 16S copy number among those organisms. Despite this fact, many studies assume that the abundance of 16S gene sequences is a surrogate measure of the relative abundance of the organisms containing those sequences. Here we present a method that uses data on sequences and genomic copy number of 16S genes along with phylogenetic placement and ancestral state estimation to estimate organismal abundances from environmental DNA sequence data. We use theory and simulations to demonstrate that 16S genomic copy number can be accurately estimated from the short reads typically obtained from high-throughput environmental sequencing of the 16S gene, and that organismal abundances in microbial communities are more strongly correlated with estimated abundances obtained from our method than with gene abundances. We re-analyze several published empirical data sets and demonstrate that the use of gene abundance versus estimated organismal abundance can lead to different inferences about community diversity and structure and the identity of the dominant taxa in microbial communities. Our approach will allow microbial ecologists to make more accurate inferences about microbial diversity and abundance based on 16S sequence data. PMID:23133348

  11. Intragenomic heterogeneity of 16S rRNA genes causes overestimation of prokaryotic diversity.

    PubMed

    Sun, Dong-Lei; Jiang, Xuan; Wu, Qinglong L; Zhou, Ning-Yi

    2013-10-01

    Ever since Carl Woese introduced the use of 16S rRNA genes for determining the phylogenetic relationships of prokaryotes, this method has been regarded as the "gold standard" in both microbial phylogeny and ecology studies. However, intragenomic heterogeneity within 16S rRNA genes has been reported in many investigations and is believed to bias the estimation of prokaryotic diversity. In the current study, 2,013 completely sequenced genomes of bacteria and archaea were analyzed and intragenomic heterogeneity was found in 952 genomes (585 species), with 87.5% of the divergence detected being below the 1% level. In particular, some extremophiles (thermophiles and halophiles) were found to harbor highly divergent 16S rRNA genes. Overestimation caused by 16S rRNA gene intragenomic heterogeneity was evaluated at different levels using the full-length and partial 16S rRNA genes usually chosen as targets for pyrosequencing. The result indicates that, at the unique level, full-length 16S rRNA genes can produce an overestimation of as much as 123.7%, while at the 3% level, an overestimation of 12.9% for the V6 region may be introduced. Further analysis showed that intragenomic heterogeneity tends to concentrate in specific positions, with the V1 and V6 regions suffering the most intragenomic heterogeneity and the V4 and V5 regions suffering the least intragenomic heterogeneity in bacteria. This is the most up-to-date overview of the diversity of 16S rRNA genes within prokaryotic genomes. It not only provides general guidance on how much overestimation can be introduced when applying 16S rRNA gene-based methods, due to its intragenomic heterogeneity, but also recommends that, for bacteria, this overestimation be minimized using primers targeting the V4 and V5 regions.

  12. Diversity of 16S rRNA Genes within Individual Prokaryotic Genomes▿ †

    PubMed Central

    Pei, Anna Y.; Oberdorf, William E.; Nossa, Carlos W.; Agarwal, Ankush; Chokshi, Pooja; Gerz, Erika A.; Jin, Zhida; Lee, Peng; Yang, Liying; Poles, Michael; Brown, Stuart M.; Sotero, Steven; DeSantis, Todd; Brodie, Eoin; Nelson, Karen; Pei, Zhiheng

    2010-01-01

    Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (http://nihroadmap.nih.gov/hmp). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 ± 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% ± 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in Haloarcula marismortui (diversity, 5.63%) and Thermoanaerobacter tengcongensis (6.70%) were highly conserved at the 2° structure level, while the diversified gene in B. afzelii (20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in “Candidatus Protochlamydia amoebophila.” Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases. PMID:20418441

  13. Diversity of 16S rRNA genes within individual prokaryotic genomes.

    PubMed

    Pei, Anna Y; Oberdorf, William E; Nossa, Carlos W; Agarwal, Ankush; Chokshi, Pooja; Gerz, Erika A; Jin, Zhida; Lee, Peng; Yang, Liying; Poles, Michael; Brown, Stuart M; Sotero, Steven; Desantis, Todd; Brodie, Eoin; Nelson, Karen; Pei, Zhiheng

    2010-06-01

    Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (http://nihroadmap.nih.gov/hmp). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 +/- 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% +/- 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in Haloarcula marismortui (diversity, 5.63%) and Thermoanaerobacter tengcongensis (6.70%) were highly conserved at the 2 degrees structure level, while the diversified gene in B. afzelii (20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in "Candidatus Protochlamydia amoebophila." Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases.

  14. Molecular identification of adulteration in mutton based on mitochondrial 16S rRNA gene.

    PubMed

    Xu, Jia; Zhao, Wei; Zhu, Mengru; Wen, Yuanju; Xie, Tao; He, Xiaoqian; Zhang, Yongfeng; Cao, Suizhong; Niu, Lili; Zhang, Hongping; Zhong, Tao

    2016-01-01

    The aim of this study is to set up a protocol for identification of the adulteration in mutton based on mitochondrial 16S rRNA gene. The multiplex polymerase chain reaction (multi-PCR) assay was carried out to trace the impure DNA in mutton. A universal primer pair yielded an approximate 610 bp fragment in mutton, pork, duck, chicken, horse and cat meats. The amplicons of multi-PCR assay represented the species-specific products, which could be discriminated by the size ranging from 106 bp to 532 bp. Subsequently, the authentication of each fragment was also confirmed by sequencing. Random analyses of adulterants with various meats yielded the identical results to their components, showing the suitability of the multi-PCR assay for tracing of adulterant meats with high-accuracy and precision. This assay was sensitive to detect the species-specific DNA in different proportional mixtures of mutton and duck/pork (9.1%-90.9%). In conclusion, this multi-PCR assay successfully discriminated the double-, triple-, quadruple-, and quintuple-mixtures containing variant counterparts. This method will be particularly useful in the detection of mutton adulteration in processed foods further.

  15. 16S rRNA Amplicon Sequencing for Epidemiological Surveys of Bacteria in Wildlife

    PubMed Central

    Razzauti, Maria; Bard, Emilie; Bernard, Maria; Brouat, Carine; Charbonnel, Nathalie; Dehne-Garcia, Alexandre; Loiseau, Anne; Tatard, Caroline; Tamisier, Lucie; Vayssier-Taussat, Muriel; Vignes, Helene

    2016-01-01

    ABSTRACT The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculus domesticus, 189 Rattus rattus, 93 Mastomys natalensis, and 221 Mastomys erythroleucus, collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia. Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial

  16. Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.)

    PubMed Central

    2012-01-01

    Background Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 are α-Proteobacteria that establish nitrogen-fixing symbioses with a range of legume hosts. These strains are broadly used in commercial inoculants for application to common bean (Phaseolus vulgaris) in South America and Africa. Both strains display intrinsic resistance to several abiotic stressful conditions such as low soil pH and high temperatures, which are common in tropical environments, and to several antimicrobials, including pesticides. The genetic determinants of these interesting characteristics remain largely unknown. Results Genome sequencing revealed that CIAT 899 and PRF 81 share a highly-conserved symbiotic plasmid (pSym) that is present also in Rhizobium leucaenae CFN 299, a rhizobium displaying a similar host range. This pSym seems to have arisen by a co-integration event between two replicons. Remarkably, three distinct nodA genes were found in the pSym, a characteristic that may contribute to the broad host range of these rhizobia. Genes for biosynthesis and modulation of plant-hormone levels were also identified in the pSym. Analysis of genes involved in stress response showed that CIAT 899 and PRF 81 are well equipped to cope with low pH, high temperatures and also with oxidative and osmotic stresses. Interestingly, the genomes of CIAT 899 and PRF 81 had large numbers of genes encoding drug-efflux systems, which may explain their high resistance to antimicrobials. Genome analysis also revealed a wide array of traits that may allow these strains to be successful rhizosphere colonizers, including surface polysaccharides, uptake transporters and catabolic enzymes for nutrients, diverse iron-acquisition systems, cell wall-degrading enzymes, type I and IV pili, and novel T1SS and T5SS secreted adhesins. Conclusions Availability of the complete genome sequences of CIAT 899 and PRF 81 may be exploited in further efforts to understand the interaction of tropical rhizobia with common bean

  17. Mixed quantum/classical calculations of total and differential elastic and rotationally inelastic scattering cross sections for light and heavy reduced masses in a broad range of collision energies

    SciTech Connect

    Semenov, Alexander; Babikov, Dmitri

    2014-01-28

    The mixed quantum/classical theory (MQCT) for rotationally inelastic scattering developed recently [A. Semenov and D. Babikov, J. Chem. Phys. 139, 174108 (2013)] is benchmarked against the full quantum calculations for two molecular systems: He + H{sub 2} and Na + N{sub 2}. This allows testing new method in the cases of light and reasonably heavy reduced masses, for small and large rotational quanta, in a broad range of collision energies and rotational excitations. The resultant collision cross sections vary through ten-orders of magnitude range of values. Both inelastic and elastic channels are considered, as well as differential (over scattering angle) cross sections. In many cases results of the mixed quantum/classical method are hard to distinguish from the full quantum results. In less favorable cases (light masses, larger quanta, and small collision energies) some deviations are observed but, even in the worst cases, they are within 25% or so. The method is computationally cheap and particularly accurate at higher energies, heavier masses, and larger densities of states. At these conditions MQCT represents a useful alternative to the standard full-quantum scattering theory.

  18. Comparative analysis of bacteria associated with different mosses by 16S rRNA and 16S rDNA sequencing.

    PubMed

    Tian, Yang; Li, Yan Hong

    2017-01-01

    To understand the differences of the bacteria associated with different mosses, a phylogenetic study of bacterial communities in three mosses was carried out based on 16S rDNA and 16S rRNA sequencing. The mosses used were Hygroamblystegium noterophilum, Entodon compressus and Grimmia montana, representing hygrophyte, shady plant and xerophyte, respectively. In total, the operational taxonomic units (OTUs), richness and diversity were different regardless of the moss species and the library level. All the examined 1183 clones were assigned to 248 OTUs, 56 genera were assigned in rDNA libraries and 23 genera were determined at the rRNA level. Proteobacteria and Bacteroidetes were considered as the most dominant phyla in all the libraries, whereas abundant Actinobacteria and Acidobacteria were detected in the rDNA library of Entodon compressus and approximately 24.7% clones were assigned to Candidate division TM7 in Grimmia montana at rRNA level. The heatmap showed the bacterial profiles derived from rRNA and rDNA were partly overlapping. However, the principle component analysis of all the profiles derived from rDNA showed sharper differences between the different mosses than that of rRNA-based profiles. This suggests that the metabolically active bacterial compositions in different mosses were more phylogenetically similar and the differences of the bacteria associated with different mosses were mainly detected at the rDNA level. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA sequencing is preferred approach to have a good understanding on the constitution of the microbial communities in mosses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns*

    PubMed Central

    Atherly, Todd; Ziemer, Cherie J

    2014-01-01

    One-hundred-and-three isolates of Bacteroides ovatus,B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade. PMID:24532571

  20. Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns.

    PubMed

    Atherly, Todd; Ziemer, Cherie J

    2014-04-01

    One-hundred-and-three isolates of Bacteroides ovatus, B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  1. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence.

    PubMed

    Hao, Huijing; Liang, Junrong; Duan, Ran; Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.

  2. Identification of grass-associated and toluene-degrading diazotrophs, Axoarcus spp., by analyses of partial 16S ribosomal DNA sequences

    SciTech Connect

    Hurek, T.; Reinhold-Hurek, B.

    1995-06-01

    The genus Azoarcus includes nitrogen-fixing, grass-associated strains as well as denitrifying toluene degraders. In order to identify and group members of the genus Azoarcus, phylogenetic analysis based on partial sequences of 16S rRNA genes (16S rDNAs) is proposed. 16S rRNA-targeted PCR using specific primers to exclude amplification in the majority of other members of the beta subclass of the class Proteobacteria was combined with direct sequencing of the PCR products. Tree inference from comparisons of 446-bp rDNA fragments yielded similar results for the three known Azoarcus spp. sequences and for analysis of the complete 16S rDNA sequence. These three species formed a phylogenetically coherent group with representatives of two other Azoarcus species which were subjected to 16S rRNA sequencing in this study. This group was related to Rhodocyclus purpureus and Thaurea selenatis. New isolates and also sequences of so far uncultured bacteria from roots of Kallar grass were assigned to the genus Azoarcus as well. Also, strains degrading monoaromatic hydrocarbons anaerobically in the presence of nitrate clustered within this genus, albeit not with grass-associated isolates. All representative members of the five species harboring rhizospheric bacteria were able to form N{sub 2}O from nitrate and showed anaerobic growth on malic acid with nitrate but not on toluene. In order to visualize different Azoarcus spp. by whole-cell in situ hybridizations, we generated 16S rRNA-targeted, fluorescent probes by in vitro transcription directly from PCR products which spanned the variable region V2. Hybridization was species specific for Azoarcus communis and Azoarcus indigens. The proposed scheme of phylogenetic analysis of PCR-generated 16S rDNA segements will facilitate studies on ecological distribution, host range, and diversity of Azoarcus spp. and may even allow rapid identification of unc ultured strains from environmental DNAs. 30 refs., 3 figs.

  3. The Human Microbiome and Understanding the 16S rRNA Gene in Translational Nursing Science.

    PubMed

    Ames, Nancy J; Ranucci, Alexandra; Moriyama, Brad; Wallen, Gwenyth R

    As more is understood regarding the human microbiome, it is increasingly important for nurse scientists and healthcare practitioners to analyze these microbial communities and their role in health and disease. 16S rRNA sequencing is a key methodology in identifying these bacterial populations that has recently transitioned from use primarily in research to having increased utility in clinical settings. The objectives of this review are to (a) describe 16S rRNA sequencing and its role in answering research questions important to nursing science; (b) provide an overview of the oral, lung, and gut microbiomes and relevant research; and (c) identify future implications for microbiome research and 16S sequencing in translational nursing science. Sequencing using the 16S rRNA gene has revolutionized research and allowed scientists to easily and reliably characterize complex bacterial communities. This type of research has recently entered the clinical setting, one of the best examples involving the use of 16S sequencing to identify resistant pathogens, thereby improving the accuracy of bacterial identification in infection control. Clinical microbiota research and related requisite methods are of particular relevance to nurse scientists-individuals uniquely positioned to utilize these techniques in future studies in clinical settings.

  4. Differential assembly of 16S rRNA domains during 30S subunit formation.

    PubMed

    Xu, Zhili; Culver, Gloria M

    2010-10-01

    Rapid and accurate assembly of the ribosomal subunits, which are responsible for protein synthesis, is required to sustain cell growth. Our best understanding of the interaction of 30S ribosomal subunit components (16S ribosomal RNA [rRNA] and 20 ribosomal proteins [r-proteins]) comes from in vitro work using Escherichia coli ribosomal components. However, detailed information regarding the essential elements involved in the assembly of 30S subunits still remains elusive. Here, we defined a set of rRNA nucleotides that are critical for the assembly of the small ribosomal subunit in E. coli. Using an RNA modification interference approach, we identified 54 nucleotides in 16S rRNA whose modification prevents the formation of a functional small ribosomal subunit. The majority of these nucleotides are located in the head and interdomain junction of the 30S subunit, suggesting that these regions are critical for small subunit assembly. In vivo analysis of specific identified sites, using engineered mutations in 16S rRNA, revealed defective protein synthesis capability, aberrant polysome profiles, and abnormal 16S rRNA processing, indicating the importance of these residues in vivo. These studies reveal that specific segments of 16S rRNA are more critical for small subunit assembly than others, and suggest a hierarchy of importance.

  5. [16S rRNA gene sequence analysis for bacterial identification in the clinical laboratory].

    PubMed

    Matsumoto, Takehisa; Sugano, Mitsutoshi

    2013-12-01

    The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. For many years, sequencing of the 16S ribosomal RNA (rRNA) gene has served as an important tool for determining phylogenetic relationships between bacteria. The features of this molecular target that make it a useful phylogenetic tool also make it useful for bacterial detection and identification in the clinical laboratory. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, and can lead to the recognition of novel pathogens and noncultured bacteria. In clinical microbiology, molecular identification based on 16S rDNA sequencing is applied fundamentally to bacteria whose identification by means of other types of techniques is impossible or difficult. However, there are some cases in which 16S rRNA gene sequence analysis can not differentiate closely related bacteria such as Shigella spp. and Escherichia coli at the species level. Thus, it is important to understand the advantages and disadvantages of 16S rRNA gene sequence analysis.

  6. Mitochondrial 16S ribosomal RNA gene for forensic identification of crocodile species.

    PubMed

    Naga Jogayya, K; Meganathan, P R; Dubey, Bhawna; Haque, I

    2013-05-01

    All crocodilians are under various threats due to over exploitation and these species have been listed in Appendix I or II of CITES. Lack of molecular techniques for the forensic identification of confiscated samples makes it difficult to enforce the law. Therefore, we herein present a molecular method developed on the basis on 16S rRNA gene of mitochondrial DNA for identification of crocodile species. We have developed a set of 16S rRNA primers for PCR based identification of crocodilian species. These novel primers amplify partial 16S rRNA sequences of six crocodile species which can be later combined to obtain a larger region (1290 bp) of 16S rRNA gene. This 16S rRNA gene could be used as an effective tool for forensic authentication of crocodiles. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these species.

  7. Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers

    PubMed Central

    Liu, Zongzhi; DeSantis, Todd Z.; Andersen, Gary L.; Knight, Rob

    2008-01-01

    The recent introduction of massively parallel pyrosequencers allows rapid, inexpensive analysis of microbial community composition using 16S ribosomal RNA (rRNA) sequences. However, a major challenge is to design a workflow so that taxonomic information can be accurately and rapidly assigned to each read, so that the composition of each community can be linked back to likely ecological roles played by members of each species, genus, family or phylum. Here, we use three large 16S rRNA datasets to test whether taxonomic information based on the full-length sequences can be recaptured by short reads that simulate the pyrosequencer outputs. We find that different taxonomic assignment methods vary radically in their ability to recapture the taxonomic information in full-length 16S rRNA sequences: most methods are sensitive to the region of the 16S rRNA gene that is targeted for sequencing, but many combinations of methods and rRNA regions produce consistent and accurate results. To process large datasets of partial 16S rRNA sequences obtained from surveys of various microbial communities, including those from human body habitats, we recommend the use of Greengenes or RDP classifier with fragments of at least 250 bases, starting from one of the primers R357, R534, R798, F343 or F517. PMID:18723574

  8. 16S ribosomal RNA sequencing and molecular serotyping of Avibacterium paragallinarum isolated from Indian field conditions.

    PubMed

    Patil, Vihang Vithalrao; Mishra, Debendranath; Mane, Dilip Vithalrao

    2017-08-01

    This study was aimed at identifying Indian field isolates of Avibacterium paragallinarum on both molecular as well as serological levels that cause infectious coryza in chickens. Species-specific polymerase chain reaction (HPG-2 PCR), and 16S ribosomal RNA (rRNA) sequencing were employed for molecular identification. Whereas, multiplex PCR technique was used for serological identification of Indian field isolates of A. paragallinarum. All three field isolates were identified as A. paragallinarum using HPG-2 PCR. The species-specific PCR results were validated using 16S rRNA sequencing. The partial 16S rRNA sequences obtained from all three isolates showed 96-99% homology with the NCBI database reference strains of A. paragallinarum. The aligned partial sequences of 16S rRNA were submitted to GenBank, and accession numbers were obtained. Multiplex PCR-based molecular serotyping showed that there are three serotypes of field isolates of A. paragallinarum, namely, strain IND101 is serovar A, strain IND102 is serovar B, and strain IND103 is serovar C. HPG-2 PCR, 16S rRNA sequencing, and multiplex PCR are proved to be more accurate, sensitive, and reliable diagnostic tools for molecular and serological identification of A. paragallinarum field isolates. These diagnostic methods can substitute conventional cultural characterization and would be much valuable to formulate quick and correct prevention and control measures against this detrimental poultry pathogen.

  9. Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad-Host-Range RP4 Conjugative Machinery ▿

    PubMed Central

    Ferrières, Lionel; Hémery, Gaëlle; Nham, Toan; Guérout, Anne-Marie; Mazel, Didier; Beloin, Christophe; Ghigo, Jean-Marc

    2010-01-01

    Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 λpir and S17-1 λpir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 λpir and S17-1 λpir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of Π-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria. PMID:20935093

  10. Design of Vibrio 16S rRNA gene specific primers and their application in the analysis of seawater Vibrio community

    NASA Astrophysics Data System (ADS)

    Liu, Yong; Yang, Guanpin; Wang, Hualei; Chen, Jixiang; Shi, Xianming; Zou, Guiwei; Wei, Qiwei; Sun, Xiuqin

    2006-04-01

    The pathogenic species of genus Vibrio cause vibriosis, one of the most prevalent diseases of maricultured animals and seafood consumers. Monitoring their kinetics in the chain of seafood production, processing and consumption is of great importance for food and mariculture safety. In order to enrich Vibrio-representing 16S ribosomal RNA gene (rDNA) fragments and identify these bacteria further real-timely and synchronously among bacterial flora in the chain, a pair of primers that selectively amplify Vibrio 16S rDNA fragments were designed with their specificities and coverage testified in the analysis of seawater Vibrio community. The specificities and coverage of two primers, VF169 and VR744, were determined theoretically among bacterial 16S rDNAs available in GenBank by using BLAST program and practically by amplifying, Vibrio 16S rDNA fragments from seawater DNA. More than 88.3% of sequences in GenBank, which showed identical matches with VR744, belong to Vibrio genus. A total of 33 clones were randomly selected and sequenced. All of the sequences showed their highest similarities to and clustered around those of diverse known Vibrio species. The primers designed are capable of retrieving a wide range of Vibrio 16S rDNA fragments specifically among bacterial flora in seawater, the most important natural environment of seafood cultivation.

  11. Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an archaebacterium

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Lanter, J. M.; Woese, C. R.

    1983-01-01

    The sequence of the 16S ribosomal RNA (rRNA) from the archaebacterium Halobacterium volcanii has been determined by DNA sequencing methods. The archaebacterial rRNA is similar to its eubacterial counterpart in secondary structure. Although it is closer in sequence to the eubacterial 16S rRNA than to the eukaryotic 16S-like rRNA, the H. volcanii sequence also shows certain points of specific similarity to its eukaryotic counterpart. Since the H. volcanii sequence is closer to both the eubacterial and the eukaryotic sequences than these two are to one another, it follows that the archaebacterial sequence resembles their common ancestral sequence more closely than does either of the other two versions.

  12. Routine Molecular Identification of Enterococci by Gene-Specific PCR and 16S Ribosomal DNA Sequencing

    PubMed Central

    Angeletti, Silvia; Lorino, Giulia; Gherardi, Giovanni; Battistoni, Fabrizio; De Cesaris, Marina; Dicuonzo, Giordano

    2001-01-01

    For 279 clinically isolated specimens identified by commercial kits as enterococci, genotypic identification was performed by two multiplex PCRs, one with ddlE. faecalis and ddlE. faecium primers and another with vanC-1 and vanC-2/3 primers, and by 16S ribosomal DNA (rDNA) sequencing. For 253 strains, phenotypic and genotypic results were the same. Multiplex PCR allowed for the identification of 13 discordant results. Six strains were not enterococci and were identified by 16S rDNA sequencing. For 5 discordant and 10 concordant enterococcal strains, 16S rDNA sequencing was needed. Because many supplementary tests are frequently necessary for phenotypic identification, the molecular approach is a good alternative. PMID:11158155

  13. Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an archaebacterium

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Lanter, J. M.; Woese, C. R.

    1983-01-01

    The sequence of the 16S ribosomal RNA (rRNA) from the archaebacterium Halobacterium volcanii has been determined by DNA sequencing methods. The archaebacterial rRNA is similar to its eubacterial counterpart in secondary structure. Although it is closer in sequence to the eubacterial 16S rRNA than to the eukaryotic 16S-like rRNA, the H. volcanii sequence also shows certain points of specific similarity to its eukaryotic counterpart. Since the H. volcanii sequence is closer to both the eubacterial and the eukaryotic sequences than these two are to one another, it follows that the archaebacterial sequence resembles their common ancestral sequence more closely than does either of the other two versions.

  14. Research Techniques Made Simple: Bacterial 16S Ribosomal RNA Gene Sequencing in Cutaneous Research.

    PubMed

    Jo, Jay-Hyun; Kennedy, Elizabeth A; Kong, Heidi H

    2016-03-01

    Skin serves as a protective barrier and also harbors numerous microorganisms collectively comprising the skin microbiome. As a result of recent advances in sequencing (next-generation sequencing), our understanding of microbial communities on skin has advanced substantially. In particular, the 16S ribosomal RNA gene sequencing technique has played an important role in efforts to identify the global communities of bacteria in healthy individuals and patients with various disorders in multiple topographical regions over the skin surface. Here, we describe basic principles, study design, and a workflow of 16S ribosomal RNA gene sequencing methodology, primarily for investigators who are not familiar with this approach. This article will also discuss some applications and challenges of 16S ribosomal RNA sequencing as well as directions for future development.

  15. Sequence of the 16S Ribosomal RNA from Halobacterium volcanii, an Archaebacterium.

    PubMed

    Gupta, R; Lanter, J M; Woese, C R

    1983-08-12

    The sequence of the 16S ribosomal RNA (rRNA) from the archaebacterium Halobacterium volcanii has been determined by DNA sequencing methods. The archaebacterial rRNA is similar to its eubacterial counterpart in secondary structure. Although it is closer in sequence to the eubacterial 16S rRNA than to the eukaryotic 16S-like rRNA, the H. volcanii sequence also shows certain points of specific similarity to its eukaryotic counterpart. Since the H. volcanii sequence is closer to both the eubacterial and the eukaryotic sequences than these two are to one another, it follows that the archaebacterial sequence resembles their common ancestral sequence more closely than does either of the other two versions.

  16. Processing pathway of Escherichia coli 16S precursor rRNA.

    PubMed Central

    Srivastava, A K; Schlessinger, D

    1989-01-01

    Immediate precursors of 16S rRNA are processed by endonucleolytic cleavage at both 5' and 3' mature termini, with the concomitant release of precursor fragments which are further metabolized by both exo- and endonucleases. In wild-type cells rapid cleavages by RNase III in precursor-specific sequences precede the subsequent formation of the mature ends; mature termini can, however, be formed directly from pre-16S rRNA with no intermediate species. The direct maturation is most evident in a strain deficient in RNase III, and the results in whole cells are consistent with results from maturation reactions in vitro. Thus, maturation does not require cleavages within the double-stranded stems that enclose mature rRNA sequences in the pre-16S rRNA. Images PMID:2646597

  17. Comprehensive Molecular Characterization of Bacterial Communities in Feces of Pet Birds Using 16S Marker Sequencing.

    PubMed

    Garcia-Mazcorro, Jose F; Castillo-Carranza, Stephany A; Guard, Blake; Gomez-Vazquez, Jose P; Dowd, Scot E; Brigthsmith, Donald J

    2017-01-01

    Birds and other animals live and evolve in close contact with millions of microorganisms (microbiota). While the avian microbiota has been well characterized in domestic poultry, the microbiota of other bird species has been less investigated. The aim of this study was to describe the fecal bacterial communities of pet birds. Pooled fecal samples from 22 flocks representing over 150 individual birds of three different species (Melopsittacus undulatus or budgerigars, Nymphicus hollandicus or cockatiels, and Serinus canaria or domestic canaries) were used for analysis using the 16S rRNA gene sequencing in the MiSeq platform (Illumina). Firmicutes was the most abundant phylum (median 88.4 %; range 12.9-98.4 %) followed by other low-abundant phyla such as Proteobacteria (median 2.3 %; 0.1-85.3 %) and Actinobacteria (median 1.7 %; 0-18.3 %). Lactobacillaceae (mostly Lactobacillus spp.) was the most abundant family (median 78.1 %; 1.4-97.5 %), especially in budgerigars and canaries, and it deserves attention because of the ascribed beneficial properties of lactic acid bacteria. Importantly, feces from birds contain intestinal, urinary, and reproductive-associated microbiota thus posing a serious problem to study one anatomical region at a time. Other groups of interest include the family Clostridiaceae that showed very low abundance (overall median <0.1 %) with the exception of two samples from cockatiels (14 and 45.9 %) and one sample from budgerigars (19.9 %). Analysis of UniFrac metrics showed that overall, the microbial communities from the 22 flocks tended to cluster together for each bird species, meaning each species shed distinctive bacterial communities in feces. This descriptive analysis provides insight into the fecal microbiota of pet birds.

  18. Genomic Insights into Geothermal Spring Community Members using a 16S Agnostic Single-Cell Approach

    NASA Astrophysics Data System (ADS)

    Bowers, R. M.

    2016-12-01

    INSTUTIONS (ALL): DOE Joint Genome Institute, Walnut Creek, CA USA. Bigelow Laboratory for Ocean Sciences, East Boothbay, ME USA. Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada. ABSTRACT BODY: With recent advances in DNA sequencing, rapid and affordable screening of single-cell genomes has become a reality. Single-cell sequencing is a multi-step process that takes advantage of any number of single-cell sorting techniques, whole genome amplification (WGA), and 16S rRNA gene based PCR screening to identify the microbes of interest prior to shotgun sequencing. However, the 16S PCR based screening step is costly and may lead to unanticipated losses of microbial diversity, as cells that do not produce a clean 16S amplicon are typically omitted from downstream shotgun sequencing. While many of the sorted cells that fail the 16S PCR step likely originate from poor quality amplified DNA, some of the cells with good WGA kinetics may instead represent bacteria or archaea with 16S genes that fail to amplify due to primer mis-matches or the presence of intervening sequences. Using cell material from Dewar Creek, a hot spring in British Columbia, we sequenced all sorted cells with good WGA kinetics irrespective of their 16S amplification success. We show that this high-throughput approach to single-cell sequencing (i) can reduce the overall cost of single-cell genome production, and (ii). may lead to the discovery of previously unknown branches on the microbial tree of life.

  19. Qualification status of hybrid crystal oscillators style OTO 16S for space application

    NASA Astrophysics Data System (ADS)

    Gerard, E.; Deviller, J. L.

    1991-03-01

    The qualification status of a crystal clock oscillator, OTO 16S, is described. Specifically designed for the Telecom 2 and Intelsat 7 programs, the oscillator is available in frequencies between 3 and 25 MHz with Transistor Transistor Logic (TTL) compatible outputs. Qualification tests results are presented to demonstrate that all the OTO 16S performances are in compliance with space requirements. From a mechanical viewpoint, no degradation is seen from a vibration level of 50 g sinus 10 to 2000 Hz. From a life test viewpoint, no significant variations are observed after 2000 hours of testing.

  20. 16S rRNA Phylogenetic Investigation of the Candidate Division “Korarchaeota”

    PubMed Central

    Auchtung, Thomas A.; Takacs-Vesbach, Cristina D.; Cavanaugh, Colleen M.

    2006-01-01

    The environmental distribution and phylogeny of “Korarchaeota,” a proposed ancient archaeal division, was investigated by using the 16S rRNA gene framework. Korarchaeota-specific primers were designed based on previously published sequences and used to screen a variety of environments. Korarchaeota 16S rRNA genes were amplified exclusively from high temperature Yellowstone National Park hot springs and a 9°N East Pacific Rise deep-sea hydrothermal vent. Phylogenetic analyses of these and all available sequences suggest that Korarchaeota exhibit a high level of endemicity. PMID:16820509

  1. Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies

    NASA Technical Reports Server (NTRS)

    Rossler, D.; Ludwig, W.; Schleifer, K. H.; Lin, C.; McGill, T. J.; Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1991-01-01

    Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

  2. Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies.

    PubMed

    Rössler, D; Ludwig, W; Schleifer, K H; Lin, C; McGill, T J; Wisotzkey, J D; Jurtshuk, P; Fox, G E

    1991-01-01

    Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

  3. [Bacterial 16S rDNA sequence analysis of Siberian tiger faecal flora].

    PubMed

    Tu, Ya; Zhu, Wei-yun; Lu, Cheng-ping

    2005-10-01

    Bacterial 16S rDNA library of Siberian tiger was developed and 15 different clones were obtained using EcoR I and Hind III in restriction fragment length polymorphism analysis. DNA sequencing and similarity analysis showed that 10 clones matched corresponding Clostridium sequences, of which 6 sequences had over 99% similarity with Clostridium novyi type A, and 4 sequences had 97% similarity with Swine manure bacterium RT-18B, which identified as Peptostreptococcus spp. The other five 16S rDNA sequences had 94% - 95% similarity with Clostridium pascui, Clostridium tetani E88, Clostridium sp. 14505 Clostridium perfringens and Carnobacterium sp. R-7279 respectively.

  4. Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies

    NASA Technical Reports Server (NTRS)

    Rossler, D.; Ludwig, W.; Schleifer, K. H.; Lin, C.; McGill, T. J.; Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1991-01-01

    Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

  5. Effect of gemini (alkanediyl-α,ω-bis(dimethylcetylammonium bromide)) (16-s-16, s=4, 5, 6) surfactants on the interaction of ninhydrin with chromium-glycylphenylalanine.

    PubMed

    Kumar, Dileep; Rub, Malik Abdul; Akram, Mohd; Kabir-ud-Din

    2014-11-11

    The effect of gemini (alkanediyl-α,ω-bis(dimethylcetylammonium bromide)) (16-s-16, s=4, 5, 6) surfactants on the interaction of ninhydrin with chromium(III) complex of glycylphenylalanine ([Cr(III)-Gly-Phe]2+) has been investigated using UV-visible spectrophotometer at different temperatures. The order of reaction with respect to [Cr(III)-Gly-Phe]2+ is unity while it is fractional with respect to ninhydrin. Whereas, the values of rate constant (kψ) increase and leveling-off regions, like conventional single chain cetyltrimethylammonium bromide (CTAB) surfactant, were observed with geminis, later produces a third region of increasing kψ at higher gemini surfactant concentrations. This unusual third-region effect of the gemini micelles is assigned to changes in their micellar morphologies. The results obtained in micellar media were treated in terms of pseudo-phase model. The values of thermodynamic parameters (Ea, ΔH# and ΔS#) and binding constants (KA and KNin) have been evaluated. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Assessment of Three Mitochondrial Genes (16S, Cytb, CO1) for Identifying Species in the Praomyini Tribe (Rodentia: Muridae)

    PubMed Central

    Nicolas, Violaine; Schaeffer, Brigitte; Missoup, Alain Didier; Kennis, Jan; Colyn, Marc; Denys, Christiane; Tatard, Caroline; Cruaud, Corinne; Laredo, Catherine

    2012-01-01

    The Praomyini tribe is one of the most diverse and abundant groups of Old World rodents. Several species are known to be involved in crop damage and in the epidemiology of several human and cattle diseases. Due to the existence of sibling species their identification is often problematic. Thus an easy, fast and accurate species identification tool is needed for non-systematicians to correctly identify Praomyini species. In this study we compare the usefulness of three genes (16S, Cytb, CO1) for identifying species of this tribe. A total of 426 specimens representing 40 species (sampled across their geographical range) were sequenced for the three genes. Nearly all of the species included in our study are monophyletic in the neighbour joining trees. The degree of intra-specific variability tends to be lower than the divergence between species, but no barcoding gap is detected. The success rate of the statistical methods of species identification is excellent (up to 99% or 100% for statistical supervised classification methods as the k-Nearest Neighbour or Random Forest). The 16S gene is 2.5 less variable than the Cytb and CO1 genes. As a result its discriminatory power is smaller. To sum up, our results suggest that using DNA markers for identifying species in the Praomyini tribe is a largely valid approach, and that the CO1 and Cytb genes are better DNA markers than the 16S gene. Our results confirm the usefulness of statistical methods such as the Random Forest and the 1-NN methods to assign a sequence to a species, even when the number of species is relatively large. Based on our NJ trees and the distribution of all intraspecific and interspecific pairwise nucleotide distances, we highlight the presence of several potentially new species within the Praomyini tribe that should be subject to corroboration assessments. PMID:22574186

  7. Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family)

    PubMed Central

    Godziszewska, Jolanta; Wojciechowska, Anna; Ludwiczak, Marta; Jagura-Burdzy, Grazyna

    2016-01-01

    The KorB protein of the broad-host-range conjugative plasmid RA3 from the IncU group belongs to the ParB family of plasmid and chromosomal segregation proteins. As a partitioning DNA-binding factor, KorB specifically recognizes a 16-bp palindrome which is an essential motif in the centromere-like sequence parSRA3, forms a segrosome, and together with its partner IncC (ParA family) participates in active DNA segregation ensuring stable plasmid maintenance. Here we show that by binding to this palindromic sequence, KorB also acts as a repressor for the adjacent mobC promoter driving expression of the mobC-nic operon, which is involved in DNA processing during conjugation. Three other promoters, one buried in the conjugative transfer module and two divergent promoters located at the border between the replication and stability regions, are regulated by KorB binding to additional KorB operators (OBs). KorB acts as a repressor at a distance, binding to OBs separated from their cognate promoters by between 46 and 1,317 nucleotides. This repressor activity is facilitated by KorB spreading along DNA, since a polymerization-deficient KorB variant with its dimerization and DNA-binding abilities intact is inactive in transcriptional repression. KorB may act as a global regulator of RA3 plasmid functions in Escherichia coli, since its overexpression in trans negatively interferes with mini-RA3 replication and stable maintenance of RA3. PMID:26850301

  8. Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

    PubMed Central

    Elbrecht, Vasco; Taberlet, Pierre; Dejean, Tony; Valentini, Alice; Usseglio-Polatera, Philippe; Beisel, Jean-Nicolas; Coissac, Eric; Boyer, Frederic

    2016-01-01

    Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate. PMID:27114891

  9. Molecular Diagnosis of Actinomadura madurae Infection by 16S rRNA Deep Sequencing

    PubMed Central

    SenGupta, Dhruba J.; Hoogestraat, Daniel R.; Cummings, Lisa A.; Bryant, Bronwyn H.; Natividad, Catherine; Thielges, Stephanie; Monsaas, Peter W.; Chau, Mimosa; Barbee, Lindley A.; Rosenthal, Christopher; Cookson, Brad T.; Hoffman, Noah G.

    2013-01-01

    Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms. PMID:24108607

  10. Molecular diagnosis of Actinomadura madurae infection by 16S rRNA deep sequencing.

    PubMed

    Salipante, Stephen J; Sengupta, Dhruba J; Hoogestraat, Daniel R; Cummings, Lisa A; Bryant, Bronwyn H; Natividad, Catherine; Thielges, Stephanie; Monsaas, Peter W; Chau, Mimosa; Barbee, Lindley A; Rosenthal, Christopher; Cookson, Brad T; Hoffman, Noah G

    2013-12-01

    Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms.

  11. 16S rRNA Phylogeny of Sponge-Associated Cyanobacteria

    PubMed Central

    Steindler, Laura; Huchon, Dorothée; Avni, Adi; Ilan, Micha

    2005-01-01

    Phylogenetic analyses of 16S rRNA sequences of sponge-associated cyanobacteria showed them to be polyphyletic, implying that they derived from multiple independent symbiotic events. Most of the symbiont sequences were affiliated to a group of Synechococcus and Prochlorococcus species. However, other symbionts were related to different groups, such as the Oscillatoriales. PMID:16000832

  12. 16S rRNA region based PCR protocol for identification and subtyping of Parvimonas micra

    PubMed Central

    Ota-Tsuzuki, C.; Brunheira, A.T.P.; Mayer, M.P.A.

    2008-01-01

    The present study established a PCR protocol in order to identify Parvimonas micra and to evaluate the intra-species diversity by PCR-RFLP of 16S rRNA partial sequence. The data indicated that the protocol was able to identify this species which could be clustered in five genotypes. PMID:24031274

  13. 16S rRNA region based PCR protocol for identification and subtyping of Parvimonas micra.

    PubMed

    Ota-Tsuzuki, C; Brunheira, A T P; Mayer, M P A

    2008-10-01

    The present study established a PCR protocol in order to identify Parvimonas micra and to evaluate the intra-species diversity by PCR-RFLP of 16S rRNA partial sequence. The data indicated that the protocol was able to identify this species which could be clustered in five genotypes.

  14. Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

    USDA-ARS?s Scientific Manuscript database

    We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Ovine foot rot is an infectious, contagious disease of sheep that causes severe lameness and economic loss from decreased flock produc...

  15. Sensitivity and correlation of hypervariable regions in 16S rRNA genes in phylogenetic analysis.

    PubMed

    Yang, Bo; Wang, Yong; Qian, Pei-Yuan

    2016-03-22

    Prokaryotic 16S ribosomal RNA (rRNA) sequences are widely used in environmental microbiology and molecular evolution as reliable markers for the taxonomic classification and phylogenetic analysis of microbes. Restricted by current sequencing techniques, the massive sequencing of 16S rRNA gene amplicons encompassing the full length of genes is not yet feasible. Thus, the selection of the most efficient hypervariable regions for phylogenetic analysis and taxonomic classification is still debated. In the present study, several bioinformatics tools were integrated to build an in silico pipeline to evaluate the phylogenetic sensitivity of the hypervariable regions compared with the corresponding full-length sequences. The correlation of seven sub-regions was inferred from the geodesic distance, a parameter that is applied to quantitatively compare the topology of different phylogenetic trees constructed using the sequences from different sub-regions. The relationship between different sub-regions based on the geodesic distance indicated that V4-V6 were the most reliable regions for representing the full-length 16S rRNA sequences in the phylogenetic analysis of most bacterial phyla, while V2 and V8 were the least reliable regions. Our results suggest that V4-V6 might be optimal sub-regions for the design of universal primers with superior phylogenetic resolution for bacterial phyla. A potential relationship between function and the evolution of 16S rRNA is also discussed.

  16. Novel essential gene Involved in 16S rRNA processing in Escherichia coli.

    PubMed

    Kurata, Tatsuaki; Nakanishi, Shinobu; Hashimoto, Masayuki; Taoka, Masato; Yamazaki, Yukiko; Isobe, Toshiaki; Kato, Jun-ichi

    2015-02-27

    Biogenesis of ribosomes is a complex process mediated by many factors. While its transcription proceeds, ribosomal RNA (rRNA) folds itself into a characteristic three-dimensional structure through interaction with ribosomal proteins, during which its ends are processed. Here, we show that the essential protein YqgF, a RuvC family protein with an RNase-H-like motif, is involved in the processing of pre-16S rRNA during ribosome maturation. Indeed, pre-16S rRNA accumulated in cells of a temperature-sensitive yqgF mutant (yqgF(ts)) cultured at a non-permissive temperature. In addition, purified YqgF was shown to process the 5' end of pre-16S rRNA within 70S ribosomes in vitro. Mass spectrometry analysis of the total proteins in the yqgF(ts) mutant cells showed that the expression of genes containing multiple Shine-Dalgarno-like sequences was observed to be lower than in wild type. These results are interpreted to indicate that YqgF is involved in a novel enzymic activity necessary for the processing of pre-16S rRNA, thereby affecting elongation of translation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Problem-Based Test: Functional Analysis of Mutant 16S rRNAs

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    Terms to be familiar with before you start to solve the test: ribosome, ribosomal subunits, antibiotics, point mutation, 16S, 5S, and 23S rRNA, Shine-Dalgarno sequence, mRNA, tRNA, palindrome, hairpin, restriction endonuclease, fMet-tRNA, peptidyl transferase, initiation, elongation, termination of translation, expression plasmid, transformation,…

  18. Problem-Based Test: Functional Analysis of Mutant 16S rRNAs

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    Terms to be familiar with before you start to solve the test: ribosome, ribosomal subunits, antibiotics, point mutation, 16S, 5S, and 23S rRNA, Shine-Dalgarno sequence, mRNA, tRNA, palindrome, hairpin, restriction endonuclease, fMet-tRNA, peptidyl transferase, initiation, elongation, termination of translation, expression plasmid, transformation,…

  19. Variability in abundance of the Bacterial and Archaeal 16S rRNA and amoA genes in water columns of northern South China Sea

    NASA Astrophysics Data System (ADS)

    Liu, H.; Yang, C.; Chen, S.; Xie, W.; Wang, P.; Zhang, C. L.

    2014-12-01

    Recent advances in marine microbial ecology have shown that ammonia-oxidizing Archaea (AOA) are more abundant than ammonia-oxidizing bacteria (AOB), although total Bacteria are more abundant than total Archaea in marine environments. This study aimed to examine the spatial distribution and abundance of planktonic archaeal and bacterial 16S rRNA- and amoA genes in the northern South China Sea. Water samples were collected at different depths at six stations (maximum depth ranging from 1800 m to 3200 m)with four stations (B2, B3, B6, B7) located along a transect from the northeastern continental slope to the Bashi Strait and the other two (D3, D5) located southwest of this transect. Quantitative PCR of the 16S rRNA- and amoA genes was used to estimate the abundances of total Archaea, total Bacteria, and AOA and AOB, respectively. At the B series stations, the abundance of bacterial 16S rRNA gene was twofold to 36fold higher than that of the archaeal 16S rRNA gene while fivefold lower to sixfold higher at the two D stations, with both genes showing peak values slightly below sea surface (5-75 m depths) at all stations. The archaeal amoA gene had similar variations with the archaeal 16S rRNA gene, but was 1-4 orders of magnitude lower than the archaeal 16S rRNA gene at all stations. Bacterial amoA gene was below the detection at all stations. Our results also show the difference in depth profiles among these stations, which may be caused by the difference in water movement between these regions. The non-detection of bacterial amoA gene indicates that ammonia-oxidizing Archaea are the dominant group of microorganisms in nitrification of the South China Sea, which is consistent with observations in other oceans.

  20. Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure.

    PubMed

    Frischer, M E; Floriani, P J; Nierzwicki-Bauer, S A

    1996-10-01

    The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [gamma-32P]ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.

  1. Riboprinting and 16S rRNA Gene Sequencing for Identification of Brewery Pediococcus Isolates

    PubMed Central

    Barney, Michael; Volgyi, Antonia; Navarro, Alfonso; Ryder, David

    2001-01-01

    A total of 46 brewery and 15 ATCC Pediococcus isolates were ribotyped using a Qualicon RiboPrinter. Of these, 41 isolates were identified as Pediococcus damnosus using EcoRI digestion. Three ATCC reference strains had patterns similar to each other and matched 17 of the brewery isolates. Six other brewing isolates were similar to ATCC 25249. The other 18 P. damnosus brewery isolates had unique patterns. Of the remaining brewing isolates, one was identified as P. parvulus, two were identified as P. acidilactici, and two were identified as unique Pediococcus species. The use of alternate restriction endonucleases indicated that PstI and PvuII could further differentiate some strains having identical EcoRI profiles. An acid-resistant P. damnosus isolate could be distinguished from non-acid-resistant varieties of the same species using PstI instead of EcoRI. 16S rRNA gene sequence analysis was compared to riboprinting for identifying pediococci. The complete 16S rRNA gene was PCR amplified and sequenced from seven brewery isolates and three ATCC references with distinctive riboprint patterns. The 16S rRNA gene sequences from six different brewery P. damnosus isolates were homologous with a high degree of similarity to the GenBank reference strain but were identical to each other and one ATCC strain with the exception of 1 bp in one strain. A slime-producing, beer spoilage isolate had 16S rRNA gene sequence homology to the P. acidilactici reference strain, in agreement with the riboprint data. Although 16S rRNA gene sequencing correctly identified the genus and species of the test Pediococcus isolates, riboprinting proved to be a better method for subspecies differentiation. PMID:11157216

  2. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons

    PubMed Central

    Fischer, Sandra; Kumar, Neeraj

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea. PMID:28097056

  3. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons.

    PubMed

    Lagkouvardos, Ilias; Fischer, Sandra; Kumar, Neeraj; Clavel, Thomas

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  4. Analysis of the Cystic Fibrosis Lung Microbiota via Serial Illumina Sequencing of Bacterial 16S rRNA Hypervariable Regions

    PubMed Central

    Diaz Caballero, Julio; Fung, Pauline; Gong, Yunchen; Donaldson, Sylva L.; Yuan, Lijie; Keshavjee, Shaf; Zhang, Yu; Yau, Yvonne C. W.; Waters, Valerie J.; Tullis, D. Elizabeth; Hwang, David M.; Guttman, David S.

    2012-01-01

    The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients. PMID:23056217

  5. Molecular analysis of 16S rRNA genes identifies potentially periodontal pathogenic bacteria and archaea in the plaque of partially erupted third molars.

    PubMed

    Mansfield, J M; Campbell, J H; Bhandari, A R; Jesionowski, A M; Vickerman, M M

    2012-07-01

    Small subunit rRNA sequencing and phylogenetic analysis were used to identify cultivable and uncultivable microorganisms present in the dental plaque of symptomatic and asymptomatic partially erupted third molars to determine the prevalence of putative periodontal pathogens in pericoronal sites. Template DNA prepared from subgingival plaque collected from partially erupted symptomatic and asymptomatic mandibular third molars and healthy incisors was used in polymerase chain reaction with broad-range oligonucleotide primers to amplify 16S rRNA bacterial and archaeal genes. Amplicons were cloned, sequenced, and compared with known nucleotide sequences in online databases to identify the microorganisms present. Two thousand three hundred two clones from the plaque of 12 patients carried bacterial sequences from 63 genera belonging to 11 phyla, including members of the uncultivable TM7, SR1, and Chloroflexi, and difficult-to-cultivate Synergistetes